Plant defensin polynucleotides and methods of use thereof

ABSTRACT

This invention relates to plant defensin polypeptides, nucleic acids encoding them, and methods of use thereof. The invention also relates to a chimeric protein containing all or a portion of the plant defensin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/178,449, filed Jun. 21, 2002, which is a continuation-in-part of U.S.application Ser. No. 10/030,516, filed Oct. 25, 2001, now abandoned,which was a national stage filing under 35 U.S.C. § 371 ofPCT/US00/11952, filed May 3, 2000, which International Application waspublished by the International Bureau in English on Nov. 16, 2000, andwhich claims the benefit of U.S. Provisional Application No. 60/133,039,filed May 7, 1999; the contents of which applications are hereinincorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid molecules thatencode plant defensins.

BACKGROUND OF THE INVENTION

Defensins are small, basic, cysteine-rich proteins that exhibit broadantipathogenic activity through the formation of multimeric pores inouter or inner biological membranes. The multimeric pores lead tomembrane disruption and depolarization. Defensins have a widephylogenetic distribution, having been found in insects, mammals, andplants.

Although plant defensins have only been identified recently and are notas well characterized as their mammalian and insect counterparts,several lines of observation suggest the importance of defensins inmediating host resistance to pathogen attack. Plant defensins have beenshown to induce a rapid K+ efflux and Ca₂+ influx in fungal hyphae aswell as alkalinization of the incubation medium. The operating mechanismhowever appears not to involve direct defensin-membrane interactions,but rather a different, possibly receptor-mediated, event (Thevissen,K., et al. (1996) J. Biol. Chem. 271:15018-15025). Defensins have alsobeen shown to accumulate systemically upon challenge by fungal pathogens(Manners, J. M., et al. (1998) Plant Mol. Biol. 38:1071-1080; Terras, F.R., et al. (1998) Planta 206:117-124; Terras, F. R., et al. (1995) PlantCell 7:573-588). Furthermore, transgenic tobacco that constitutivelyexpressed a radish defensin was found to have improved resistance toinfection by a fungal pathogen (Terras, F. R et al., (1995) Plant Cell7:573-588).

Defensins have been shown to be induced by artificial drought (Maitra,N. and Cushman, J. C. (1998) Plant Physiol. 118:1536) and salt stress(Yamada, S., et al. (1997) Plant Physiol. 115:314) suggesting that theseproteins may play a more general role in stress tolerance, one that isnot restricted to pathogen attack.

Defensin molecules may be used in transgenic plants in order to produceplants with increased resistance to pathogens such as fungi, viruses,bacteria, nematodes, and insects. Thus, the present invention solvesneeds for the enhancement of a plant's defensive response via amolecularly based mechanism that can be quickly incorporated intocommercial crops.

SUMMARY OF THE INVENTION

Compositions and methods relating to pathogen resistance are provided.

The defensin sequences of the present invention find use in enhancingthe plant pathogen defense system. The compositions and methods of theinvention can be used for enhancing resistance to plant pathogensincluding fungal pathogens, plant viruses, microorganisms, nematodes,insects, and the like. The method involves stably transforming a plantwith a nucleotide sequence capable of modulating the plant pathogendefense system operably linked with a promoter capable of drivingexpression of a gene in a plant cell. The defensin sequencesadditionally find use in manipulating these processes in transformedplants and plant cells.

Transformed plants, plant cells, and seeds, as well as methods formaking such plants, plant cells, and seeds, are additionally provided.It is recognized that a variety of promoters will be useful in theinvention, the choice of which will depend in part upon the desiredlevel of expression of the disclosed defensin sequences. It isrecognized that the levels of expression can be controlled to modulatethe levels of expression in the plant cell.

The present invention concerns isolated polynucleotides comprising anucleotide sequence selected from the group consisting of: a nucleotidesequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 46, or 48; a nucleotide sequence that encodes apolypeptide having the amino acid sequence set forth in SEQ ID NO: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 32, 47, or 49; anucleotide sequence that encodes a mature polypeptide having the aminoacid sequence set forth in SEQ ID NO: 35; a nucleotide sequencecharacterized by at least 75% sequence identity to the nucleotidesequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 46, or 48; a nucleotide sequence characterized by atleast 80% sequence identity to the nucleotide sequence set forth in SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 46, or48; a nucleotide sequence characterized by at least 85% sequenceidentity to the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 46, or 48; a nucleotidesequence characterized by at least 90% sequence identity to thenucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 46, or 48; and a nucleotide sequencethat comprises the complement of any one of the above.

In a further embodiment the isolated polynucleotide of the claimedinvention comprises a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 46, or 48, that codes for the polypeptide selected from thegroup consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 47, or 49.

This invention also relates to a chimeric gene comprising an isolatedpolynucleotide of the present invention operably linked to suitableregulatory sequences.

In yet a further embodiment, the present invention concerns an isolatedhost cell comprising a chimeric gene of the present invention or anisolated polynucleotide of the present invention. The host cell may beeukaryotic, such as a yeast or a plant cell, or prokaryotic, such as abacterial cell. The present invention also relates to a virus,preferably a baculovirus, comprising an isolated polynucleotide of thepresent invention or a chimeric gene of the present invention.

The present invention further provides a process for producing anisolated host cell comprising a chimeric gene of the present inventionor an isolated polynucleotide of the present invention, the processcomprising either transforming or transfecting an isolated compatiblehost cell with a chimeric gene or isolated polynucleotide of the presentinvention.

The present invention also provides an isolated polypeptide selectedfrom the group consisting of: a polypeptide comprising an amino acidsequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 32, 47, or 49; a polypeptide characterized by at least 80%identity to SEQ ID NO: 6, 26, or 28; a polypeptide characterized by atleast 85% identity to SEQ ID NO: 8; a polypeptide characterized by atleast 95% identity to SEQ ID NO: 2, 4, 10, 12, 14, 16, 18, 20, 22, 24,or 47; a polypeptide characterized by at least 97% identity to SEQ IDNO: 32; and the polypeptide of SEQ ID NO: 49.

In a further embodiment, the invention relates to a method of selectingan isolated polynucleotide that affects the level of expression of aplant defensin polypeptide or enzyme activity in a host cell, preferablya plant cell, the method comprising the steps of: (a) constructing anisolated polynucleotide of the present invention or an isolated chimericgene of the present invention; (b) introducing the isolatedpolynucleotide or the isolated chimeric gene into a host cell; (c)measuring the level of the plant defensin polypeptide or enzyme activityin the host cell containing the isolated polynucleotide; and (d)comparing the level of the plant defensin polypeptide or enzyme activityin the host cell containing the isolated polynucleotide with the levelof the plant defensin polypeptide or enzyme activity in the host cellthat does not contain the isolated polynucleotide.

A method for impacting a plant pathogen comprising introducing into aplant or cell thereof at least one nucleotide construct comprising anucleotide sequence operably linked to a promoter that drives expressionof a gene in plant cells, wherein said nucleotide sequence is selectedfrom the group consisting of: a nucleotide sequence set forth in SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 46, or48; a nucleotide sequence that encodes a polypeptide having the aminoacid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 32, 47, or 49; a nucleotide sequence that encodes amature polypeptide having the amino acid sequence set forth in SEQ IDNO: 35; a nucleotide sequence characterized by at least 75% sequenceidentity to the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 46, or 48; a nucleotidesequence characterized by at least 80% sequence identity to thenucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 46, or 48; a nucleotide sequencecharacterized by at least 85% sequence identity to the nucleotidesequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 46, or 48; a nucleotide sequence characterized by atleast 90% sequence identity to the nucleotide sequence set forth in SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 46, or48; and a nucleotide sequence that comprises the complement of any oneof the above is also provided.

Expression cassettes and stably transformed plants comprising one ormore of the defensin sequences of the invention are also provided. Thepolypeptides of the present invention are useful in protecting plantsfrom various pests including, but not limited to, insects, fungi, andnematodes.

The invention provides nucleic acid molecules comprising nucleotidesequences, and fragments and variants thereof, that encode polypeptidesor mature polypeptides that possess activity against plant pathogens. Insome embodiments, the nucleotide sequences encode polypeptides that arepesticidal against nematodes. In other embodiments, the nucleotidesequences encode polypeptides that are active against fungal pathogens.

In a particular embodiment, a transformed plant of the invention can beproduced using a defensin nucleotide sequence of the invention that hasbeen optimized for increased expression in a host plant. For example,the defensin-like polypeptides of the invention can be back-translatedto produce nucleic acids comprising codons optimized for expression in aparticular host, for example a crop plant such as a soybean plant. Insome embodiments, the invention provides transgenic plants expressingpolypeptides that find use in methods for impacting various plantpathogens.

BRIEF DESCRIPTION OF THE DRAWING AND SEQUENCE DESCRIPTIONS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing that forma part of this application.

FIG. 1 depicts the amino acid sequence alignment between the defensinencoded by the nucleotide sequences derived from Dimorphotheca sinuataclone dms2c.pk001.d3 (SEQ ID NO: 4); Picramnia pentandra clonepps.pk0011.a9 (SEQ ID NO: 8); Parthenium argentatum Grey clonesepb1c.pk002.h2 (SEQ ID NO: 12), epblc.pk001.h15 (SEQ ID NO: 14),epblc.pk003.p14 (SEQ ID NO: 16), epblc.pk004.p22 (SEQ ID NO: 18),epblc.pk005.o6 (SEQ ID NO: 20), epblc.pk006.k15 (SEQ ID NO: 22), andepb3c.pk009j22 (SEQ ID NO: 24); and Nicotiana benthamiana clonetdr1c.pk002.g7 (SEQ ID NO:28); and the defensin polypeptide isolatedfrom Dahlia merckii (NCBI GenBank Identifier (GI) No. 2147320; SEQ IDNO:33). Amino acids that are conserved among all and at least twosequences with an amino acid at that position are indicated with anasterisk (*). Dashes are used by the program to maximize alignment ofthe sequences.

FIG. 2 depicts the amino acid sequence alignment between the defensinencoded by the nucleotide sequences derived from Picramnia pentandraclone pps.pk0010.g2, also referred to as Pps-AMP1, (SEQ ID NO: 30);Vernonia mespilifolia clone vs1n.pk0009.h6 (SEQ ID NO: 32); Helianthusannuus clone hsslc.pk018.k14 (SEQ ID NO: 47); Vernonia mespilifoliaclone vsln.pk007.a9 (SEQ ID NO: 49); and the defensin polypeptideisolated from Dahlia merckii (NCBI GenBank Identifier (GI) No. 2147320;SEQ ID NO: 33). Amino acids that are conserved among each of thesequences shown are identified in the consensus sequence (represented asSEQ ID NO: 51 in the sequence listing). Dots are used by the program tomaximize alignment of the sequences. The alignment shown in FIG. 2 wasgenerated by the PILEUP program available in the Wisconsin GeneticsSoftware Package (available from Genetics Computer Group (GCG), 575Science Drive, Madison, Wis. USA).

FIG. 3 depicts the results obtained from Soybean Cyst Nematode (SCN;Heterodera glycines) assays on soybean plants transformed with a vectorcomprising the UCP1 promoter, the Barley Alpha Amylase (BAA) signalsequence, and the mature peptide region of the clone designatedpps.pk0010.g2, also known as Pps-AMP1. Average number of cysts for theJack soybean cultivar representing T0 transformants that did not containthe heterologous DNA (Neg control); the Essex soybean variety 9 which issusceptible to SCN); and selected PCR positive T0 UCP1:BAA-maturePpsAMP1 transformants. Measurements are based on 3 plants/event.

FIG. 4 depicts the results obtained from SCN assays on soybean plantstransformed with a vector comprising the IFS 1 promoter, the BAA signalsequence, and the mature peptide region of Pps-AMP1. Average number ofcysts for the Jack soybean cultivar representing T0 transformants thatdid not contain the heterologous DNA (Neg control); the Essex soybeanvariety (which is susceptible to SCN); and selected PCR positive T0IFS1:BAA-mature PpsAMP1 transformants. Measurements are based on 3plants/event.

FIG. 5 depicts the results obtained from Sclerotinia sclerotiorum leafassays on soybean plants transformed with a vector comprising the UCP1promoter, the BAA signal sequence and the mature peptide region ofPps-AMP1. Average lesion size for Essex soybean leaves (a varietysusceptible to SCN); Jack soybean cultivar leaves representing T0transformants that did not contain the heterologous DNA (Neg control);and selected transgenic UCP1:BAA-mature Pps-AMP1 T0 plants. Measurementsare based on 3 plants/event, 2 leaves/plant. Two measurements were madeacross the widest tranects of the lesion and then multiplied to give anapproximate surfact area for thelesion.

FIG. 6 depicts the results obtained from Sclerotinia sclerotiorum leafassays on soybean plants transformed with a vector comprising the IFS1promoter, the BAA signal sequence, and the mature peptide region ofPps-AMP1. Average lesion size for control Essex soybean leaves andselected transgenic IFS 1:BAA-mature PpsAMP1 T0 plants. Measurements arebased on 3 plants/event, 2 leaves/plant. Two measurements were madeacross the widest transects of the lesion and then multiplied to give anapproximate surface area for the lesion.

Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. Table 1 also identifies the cDNA clonesas individual ESTs (“EST”), the sequences of the entire cDNA insertscomprising the indicated cDNA clones (“FIS”), contigs assembled from twoor more ESTs (“Contig”), contigs assembled from an FIS and one or moreESTs (“Contig*”), or sequences encoding at a minimum the mature proteinderived from an EST, FIS, a contig, or an FIS and PCR (“CGS”).Nucleotide SEQ ID NOs: 1, 5, 9, and 25 correspond to nucleotide SEQ IDNOs: 1, 5, 3, and 7, respectively, presented in U.S. ProvisionalApplication No. 60/133,039, filed May 7, 1999. Amino acid SEQ ID NOs: 2,6, 10, and 26 correspond to amino acid SEQ ID NOs: 2, 6, 4, and 8,respectively, presented in U.S. Provisional Application No. 60/133,039,filed May 7, 1999. The sequence descriptions and Sequence Listingattached hereto comply with the rules governing nucleotide and/or aminoacid sequence disclosures in patent applications as set forth in 37C.F.R. §1.821-1.825. TABLE 1 Plant Defensins Protein SEQ ID NO: SEQ IDNO: (Plant Source) Clone Designation Status (Nucleotide) (Amino Acid)Plant Defensin (Dimorphotheca dms2c.pk001.d3 EST 1 2 sinuata) PlantDefensin (Dimorphotheca dms2c.pk001.d3(FIS) CGS 3 4 sinuata) PlantDefensin pps.pk0011.a9 EST 5 6 (Picramnia pentandra) Plant Defensinpps.pk0011.a9(FIS) CGS 7 8 (Picramnia pentandra) Plant Defensinepb1c.pk002.h2(EST) CGS 9 10 (Parthenium argentatum Grey) Plant Defensinepb1c.pk002.h2(FIS) CGS 11 12 (Parthenium argentatum Grey) PlantDefensin epb1c.pk001.h15(EST) CGS 13 14 (Parthenium argentatum Grey)Plant Defensin epb1c.pk003.p14(EST) CGS 15 16 (Parthenium argentatumGrey) Plant Defensin epb1c.pk004.p22(EST) CGS 17 18 (Partheniumargentatum Grey) Plant Defensin epb1c.pk005.o6(EST) CGS 19 20(Parthenium argentatum Grey) Plant Defensin epb1c.pk006.k15(EST) CGS 2122 (Parthenium argentatum Grey) Plant Defensin epb3c.pk009.j22(EST) CGS23 24 (Parthenium argentatum Grey) Plant Defensin tdr1c.pk002.g7(EST)CGS 25 26 (Nicotiana benthamiana) Plant Defensin tdr1c.pk002.g7(FIS) CGS27 28 (Nicotiana benthamiana) Plant Defensin pps.pk0010.g2 (FIS) CGS 2930 (Picramnia pentandra) Plant Defensin vs1n.pk0009.h6 (FIS) CGS 31 32(Vernonia mespilifolia) Plant Defensin hss1c.pk018.k14(FIS) CGS 46 47(Helianthus annuus) Plant Defensin vs1n.pk007.a9(FIS) CGS 48 49(Vernonia mespilifolia)

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984), which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, inter alia, compositions and methods formodulating the total level of polypeptides of the present inventionand/or altering their ratios in a plant. By “modulation” an increase ordecrease in a particular character, quality, substance, or response isintended. The compositions comprise nucleotide and amino acid sequencefrom various plant species.

By “plant defensin genes”, is intended genes that are structurallyrelated to plant defensins, and include thionins, small cysteine-richpeptides, proteinase inhibitors, amylase inhibitors, and the like. Theyare called defensin genes after a structural classification of proteins(SCOP) classification system. Defensins play a role in defense, morespecifically plant defense against pathogens, and they share similarityin primary and secondary structure with insect defensins. While notbound by any mechanism of action, expression of the sequences andrelated genes around disease-induced lesions may control symptomdevelopment, as in a hypersensitive response (HR), by controlling theprotease-mediated cell death mechanism. The compositions may alsofunction directly as antipathogenic proteins by inhibiting proteasesproduced by pathogens or by binding cell wall components of pathogens.Thirdly, they may also act as amphipathic proteins that perturb membranefunction, leading to cellular toxicity of the pathogens. These smallcysteine-rich peptides demonstrate antimicrobial activity. By“antimicrobial” or “antimicrobial activity” is intended antibacterial,antiviral, antinematocidal, insecticidal, and antifungal activity.Accordingly, the polypeptides of the invention may enhance resistance toinsects and nematodes. Any one defensin exhibits a spectrum ofantimicrobial activity that may involve one or more antibacterial,antifungal, antiviral, insecticidal, antinematocidal, or antipathogenicactivities. They may also be useful in regulating seed storage proteinturnover and metabolism.

Plant defensins generally comprise about 45-54 amino acids with fourdisulfide bridges (Broekaert et al. (1995) Plant Physiol. (Bethesda)108:1353-1358). The defensin genes of the present invention find use inenhancing the plant pathogen defense system. The defensins of theinvention inhibit the growth of a broad range of pathogens, includingbut not limited to fungi, bacteria, nematodes, insects, and viruses, atmicromolar concentrations. Thus, by “defensin-like activity” it isintended that the peptides inhibit pathogen growth or damage caused by avariety of pathogens, including, but not limited to, fungi, insects,nematodes, viruses, and bacteria. Defensins inhibit pathogen damagethrough a variety of mechanisms including, but not limited to,alteration of membrane ion permeability and induction of hyphalbranching in fungal targets (Garcia-Olmeda et al. (1998) Biopolymers,Peptide Science 47:479-491, herein incorporated by reference).

The compositions of the invention can be used in a variety of methodswhereby the protein products can be expressed in crop plants to functionas antimicrobial proteins. The compositions of the invention may beexpressed in a crop plant such as the soybean to function as anantifungal agent, an antinematocidal agent, and the like. Expression ofthe proteins of the invention will result in alterations or modulationof the level, tissue, or timing of expression to achieve enhanceddisease, insect, nematode, viral, fungal, or stress resistance. Thecompositions of the invention may be expressed in the native speciesincluding, but not limited to Dimorphotheca sinuata, Picramniapentandra, Parthenium argentatum Grey, Nicotiana benthamiana, Vernoniamespilifolia, and Helianthus annuus, or alternatively, can beheterologously expressed in any plant of interest. In this manner, thecoding sequence for the defensin can be used in combination with apromoter that is introduced into a crop plant. In one embodiment, ahigh-level expressing constitutive promoter may be utilized and wouldresult in high levels of expression of the defensin. In otherembodiments, the coding sequence may be operably linked to atissue-preferred promoter to direct the expression to a plant tissueknown to be susceptible to a pathogen. Likewise, manipulation of thetiming of expression may be utilized. For example, by judicious choiceof promoter, expression can be enhanced early in plant growth to primethe plant to be responsive to pathogen attack. Likewise, pathogeninducible promoters can be used wherein expression of the defensin isturned on in the presence of the pathogen.

If desired, a transit peptide can be utilized to direct cellularlocalization of the protein product. In this manner, the native transitpeptide or a heterologous transit peptide can be used. However, it isrecognized that both extracellular expression and intracellularexpression are encompassed by the methods of the invention.

Sequences of the invention, as discussed in more detail below, encompasscoding sequences, antisense sequences, and fragments and variantsthereof. Expression of the sequences of the invention can be used tomodulate or regulate the expression of corresponding defensin proteins.

The compositions and methods of the invention can be used for enhancingresistance to plant pathogens including fuigal pathogens, plant viruses,insect pathogens, bacterial pathogens, nematodes, and the like. Themethod involves stably transforming a plant with a nucleotide sequencecapable of modulating the plant pathogen defense system operably linkedwith a promoter capable of driving expression of a gene in a plant cell.By “enhancing resistance” increasing the tolerance of the plant topathogens is intended. That is, the defensin may slow or preventpathogen infection and/or spread.

In the context of this disclosure, a number of terms shall be utilized.The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acidsequence”, and “nucleic acid fragment”/“isolated nucleic acid fragment”are used interchangeably herein. These terms encompass nucleotidesequences and the like. A polynucleotide may be a polymer of RNA or DNAthat is single- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, synthetic DNA, or mixtures thereof. An isolatedpolynucleotide of the present invention may include at least one of 60contiguous nucleotides, preferably at least one of 40 contiguousnucleotides, most preferably one of at least 30 contiguous nucleotidesderived from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 46, or 48, or the complement of such sequences.

The term “isolated” polynucleotide refers to a polynucleotide that issubstantially free from other nucleic acid sequences, such as otherchromosomal and extrachromosomal DNA and RNA, that normally accompany orinteract with it as found in its naturally occurring environment.Isolated polynucleotides may be purified from a host cell in which theynaturally occur. Conventional nucleic acid purification methods known toskilled artisans may be used to obtain isolated polynucleotides. Theterm also embraces recombinant polynucleotides and chemicallysynthesized polynucleotides.

The term “recombinant” means, for example, that a nucleic acid sequenceis made by an artificial combination of two otherwise separated segmentsof sequence, e.g., by chemical synthesis or by the manipulation ofisolated nucleic acids by genetic engineering techniques.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to nucleic acid fragments wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by gene silencingthrough, for example, antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidfragments of the instant invention such as deletion or insertion of oneor more nucleotides that do not substantially affect the functionalproperties of the resulting transcript vis-à-vis the ability to mediategene silencing or alteration of the functional properties of theresulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary nucleotide oramino acid sequences and includes functional equivalents thereof. Theterms “substantially similar” and “corresponding substantially” are usedinterchangeably herein.

Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a plant or plantcell. For example, a substantially similar nucleic acid fragmentrepresenting at least one of 30 contiguous nucleotides derived from theinstant nucleic acid fragment can be constructed and introduced into aplant or plant cell. The level of the polypeptide encoded by theunmodified nucleic acid fragment present in a plant or plant cellexposed to the substantially similar nucleic fragment can then becompared to the level of the polypeptide in a plant or plant cell thatis not exposed to the substantially similar nucleic acid fragment.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby nucleic acid fragments that do not share 100% sequence identity withthe gene to be suppressed. Moreover, alterations in a nucleic acidfragment that result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts. Consequently, an isolated polynucleotide comprising anucleotide sequence of at least one of 60 (preferably at least one of40, most preferably at least one of 30) contiguous nucleotides derivedfrom a nucleotide sequence selected from the group consisting of SEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 46, or48, and the complement of such nucleotide sequences may be used inmethods of selecting an isolated polynucleotide that affects theexpression of a plant defensin polypeptide in a host cell. A method ofselecting an isolated polynucleotide that affects the level ofexpression of a polypeptide in a virus or in a host cell (eukaryotic,such as plant or yeast, prokaryotic such as bacterial) may comprise thesteps of: constructing an isolated polynucleotide of the presentinvention or an isolated chimeric gene of the present invention;introducing the isolated polynucleotide or the isolated chimeric geneinto a host cell; measuring the level of a polypeptide or enzymeactivity in the host cell containing the isolated polynucleotide; andcomparing the level of a polypeptide or enzyme activity in the host cellcontaining the isolated polynucleotide with the level of a polypeptideor enzyme activity in a host cell that does not contain the isolatedpolynucleotide.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by either DNA-DNA or DNA-RNA hybridization under conditionsof stringency as is well understood by those skilled in the art (Hamesand Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,U.K.). Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the Tm can be approximated from theequation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:Tm=81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is themolarity of monovalent cations, % GC is the percentage of guanosine andcytosine nucleotides in the DNA, % form is the percentage of formamidein the hybridization solution, and L is the length of the hybrid in basepairs. The Tm is the temperature (under defined ionic strength and pH)at which 50% of a complementary target sequence hybridizes to aperfectly matched probe. Tm is reduced by about 1° C. for each 1% ofmismatching; thus, Tm, hybridization, and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with >90% identity are sought, the Tm can be decreased 10°C. Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Tm) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3,or 4° C. lower than the thermal melting point (Tm); moderately stringentconditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10°C. lower than the thermal melting point (Tm); low stringency conditionscan utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C.lower than the thermal melting point (Tm). Using the equation,hybridization and wash compositions, and desired Tm, those of ordinaryskill will understand that variations in the stringency of hybridizationand/or wash solutions are inherently described. If the desired degree ofmismatching results in a Tm of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y.).

One set of preferred conditions uses a series of washes starting with6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set ofstringent conditions uses higher temperatures in which the washes areidentical to those above except for the temperature of the final two 30min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Anotherpreferred set of highly stringent conditions uses two final washes in0.1×SSC, 0.1% SDS at 65° C.

Thus, isolated sequences that encode a defensin polypeptide and whichhybridize under stringent conditions to the defensin sequences disclosedherein, or to fragments thereof, are encompassed by the presentinvention.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local homology algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the homology alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-similarity-method of Pearson and Lipman (1988) Proc. Natl.Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990)Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul(1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters.

The GAP program uses the algorithm of Needleman and Wunsch (1970) J.Mol. Biol. 48:443-453, to find the alignment of two complete sequencesthat maximizes the number of matches and minimizes the number of gaps.GAP considers all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. Default gap creationpenalty values and gap extension penalty values in Version 10 of theWisconsin Genetics Software Package for protein sequences are 8 and 2,respectively. For nucleotide sequences the default gap creation penaltyis 50 while the default gap extension penalty is 3. The gap creation andgap extension penalties can be expressed as an integer selected from thegroup of integers consisting of from 0 to 200. Thus, for example, thegap creation and gap extension penalties can each be 0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.The scoring matrix used in Version 10 of the Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

The CLUSTAL program is well described by Higgins et al. (1988) Gene73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al.(1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. TheALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences.

The BLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403 arebased on the algorithm of Karlin and Altschul (1990) supra. BLASTnucleotide searches can be performed with the BLASTN program, score=100,wordlength=12, to obtain nucleotide sequences homologous to a nucleotidesequence encoding a protein of the invention. BLAST protein searches canbe performed with the BLASTX program, score=50, wordlength=3, to obtainamino acid sequences homologous to a protein or polypeptide of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST (in BLAST 2.0) can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST2.0) can be used to perform an iterated search that detects distantrelationships between molecules. See Altschul et al. (1997) supra. Whenutilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of therespective programs (e.g., BLASTN for nucleotide sequences, BLASTX forproteins) can be used. Alignment may also be performed manually byinspection.

Unless otherwise indicated, sequence alignments and percent identitycalculations were performed using the Megalign program of the LASERGENEbioinformatics computing suite (DNASTAR Inc., Madison, Wis.), or anyequivalent program. Multiple alignment of the sequences was performedusing the Clustal method of alignment (Higgins and Sharp (1989) CABIOS5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTHPENALTY=10), while default parameters for pairwise alignments using theClustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5, unless otherwise indicated.

By “equivalent program” any sequence comparison program that, for anytwo sequences in question, generates an alignment having identicalnucleotide or amino acid residue matches and an identical percentsequence identity when compared to the corresponding alignment generatedby the preferred program is intended.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences makes reference to the residues inthe two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

As used herein, “comparison window” makes reference to a contiguous andspecified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e, gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence, a gap penalty is typically introduced and is subtracted fromthe number of matches.

As used herein, “full-length sequence” in reference to a specifiedpolynucleotide or its encoded protein means having the entire nucleicacid sequence or the entire amino acid sequence of a native(non-synthetic), endogenous sequence. A full-length polynucleotideencodes the full-length, catalytically active form of the specifiedprotein.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 70% sequenceidentity, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identitycompared to a reference sequence using one of the alignment programsdescribed using standard parameters. One of skill in the art willrecognize that these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleotide sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning, and the like. Substantial identity of amino acidsequences for these purposes normally means sequence identity of atleast 60%, more preferably at least 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, and 99%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. However, stringent conditions encompasstemperatures in the range of about 1° C. to about 20° C., depending uponthe desired degree of stringency as otherwise qualified herein. Nucleicacids that do not hybridize to each other under stringent conditions arestill substantially identical if the polypeptides they encode aresubstantially identical. This may occur, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code. One indication that two nucleic acid sequences aresubstantially identical is when the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

The term “substantial identity” in the context of a peptide indicatesthat a peptide comprises a sequence with at least 70% sequence identityto a reference sequence, preferably 80%, more preferably 85%, mostpreferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to the reference sequence over a specified comparisonwindow. Preferably, optimal alignment is conducted using the homologyalignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443. Anindication that two peptide sequences are substantially identical isthat one peptide is immunologically reactive with antibodies raisedagainst the second peptide. Thus, a peptide is substantially identicalto a second peptide, for example, where the two peptides differ only bya conservative substitution. Peptides that are “substantially similar”share sequences as noted above except that residue positions that arenot identical may differ by conservative amino acid changes.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410). In general, asequence of ten or more contiguous amino acids or thirty or morecontiguous nucleotides is necessary in order to putatively identify apolypeptide or nucleic acid sequence as homologous to a known protein orgene. Moreover, with respect to nucleotide sequences, gene-specificoligonucleotide probes comprising 30 or more contiguous nucleotides maybe used in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12 or more nucleotides may be used as amplificationprimers in PCR in order to obtain a particular nucleic acid fragmentcomprising the primers. Accordingly, a “substantial portion” of anucleotide sequence comprises a nucleotide sequence that will affordspecific identification and/or isolation of a nucleic acid fragmentcomprising the sequence. The instant specification teaches amino acidand nucleotide sequences encoding polypeptides that comprise one or moreparticular plant proteins. The skilled artisan, having the benefit ofthe sequences as reported herein, may now use all or a substantialportion of the disclosed sequences for purposes known to those skilledin this art. Accordingly, the instant invention comprises the completesequences as reported in the accompanying Sequence Listing, as well assubstantial portions of those sequences as defined above.

Fragments and variants of the disclosed nucleotide sequences andproteins encoded thereby are also encompassed by the present invention.By “fragment” a portion of the nucleotide sequence or a portion of theamino acid sequence and hence protein encoded thereby is intended.Fragments of a nucleotide sequence may encode protein fragments thatretain the biological activity of the native protein and hence havedefensin-like activity and thereby affect development, developmentalpathways, and defense responses. Alternatively, fragments of anucleotide sequence that are useful as hybridization probes generally donot encode fragment proteins retaining biological activity. Thus,fragments of a nucleotide sequence may range from at least about 20nucleotides, about 50 nucleotides, about 100 nucleotides, and up to thefull-length nucleotide sequence encoding the proteins of the invention.

A fragment of a defensin nucleotide sequence that encodes a biologicallyactive portion of a defensin protein of the invention will encode atleast 15, 25, 30, 50, 100, 150, 153, 200, 250, 300, contiguous aminoacids, or up to the total number of amino acids present in a full-lengthprotein of the invention. Fragments of a defensin nucleotide sequencethat are useful as hybridization probes for PCR primers generally neednot encode a biologically active portion of a defensin protein.

Thus, a fragment of a defensin nucleotide sequence may encode abiologically active portion of a defensin protein, or it may be afragment that can be used as a hybridization probe or PCR primer usingmethods disclosed below. A biologically active portion of a defensinprotein can be prepared by isolating a portion of one of the defensinnucleotide sequences of the invention, expressing the encoded portion ofthe defensin protein (e.g., by recombinant expression in vitro), andassessing the activity of the encoded portion of the defensin protein.Nucleic acid molecules that are fragments of a defensin nucleotidesequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 683, 700, 800, or 900 nucleotides, or upto the number of nucleotides present in a full-length defensinnucleotide sequence disclosed herein.

Biological activity of the defensin polypeptides (i.e., influencing theplant defense response and various developmental pathways, including,for example, influencing cell division) can be assayed by any methodknown in the art (see for example, U.S. Pat. No. 5,614,395; Thomma etal. (1998) Plant Biology 95:15107-15111; Liu et al. (1994) Plant Biology91:1888-1892; Hu et al. (1997) Plant Mol. Biol. 34:949-959; Cammue etal. (1992) J. Biol. Chem. 267: 2228-2233; and Thevissen et al. (1996) J.Biol. Chem. 271:15018-15025, all of which are herein incorporated byreference). Furthermore, assays to detect defensin-like activityinclude, for example, assessing antifungal and/or antimicrobial activity(Terras et al. (1992) J. Biol. Chem. 267:14301-15309; Terras et al.(1993) Plant Physiol (Bethesda) 103:1311-1319; Terras et al. (1995)Plant Cell 7:573-588, Moreno et al. (1994) Eur. J. Biochem. 223:135-139;and Osborn et al. (1995) FEBS Lett. 368:257-262, all of which are hereinincorporated by reference).

By “variants” substantially similar sequences are intended. Fornucleotide sequences, conservative variants include those sequencesthat, because of the degeneracy of the genetic code, encode the aminoacid sequence of one of the defensin polypeptides of the invention.Naturally occurring allelic variants such as these can be identifiedwith the use of well-known molecular biology techniques, as, forexample, with polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant nucleotide sequences also includesynthetically derived nucleotide sequences, such as those generated, forexample, by using site-directed mutagenesis but which still encode adefensin protein of the invention. Generally, variants of a particularnucleotide sequence of the invention will have at least about 50%, 60%,65%, 70%, generally at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, preferably at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, and more preferably at least about 98%, 99% or moresequence identity to that particular nucleotide sequence as determinedby sequence alignment programs described elsewhere herein using defaultparameters.

By “variant protein” a protein derived from the native protein bydeletion (so-called truncation) or addition of one or more amino acidsto the N-terminal and/or C-terminal end of the native protein; deletionor addition of one or more amino acids at one or more sites in thenative protein; or substitution of one or more amino acids at one ormore sites in the native protein is intended. Variant proteinsencompassed by the present invention are biologically active, that isthey continue to possess the desired biological activity of the nativeprotein, that is, defensin-like activity as described herein. Suchvariants may result from, for example, genetic polymorphism or fromhuman manipulation. Biologically active variants of a native defensinprotein of the invention will have at least about 40%, 50%, 60%, 65%,70%, generally at least about 75%, 80%, 85%, preferably at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and more preferably at leastabout 98%, 99% or more sequence identity to the amino acid sequence forthe native protein as determined by sequence alignment programsdescribed elsewhere herein using default parameters. A biologicallyactive variant of a protein of the invention may differ from thatprotein by as few as 1-15 amino acid residues, as few as 1-10, such as6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

The polypeptides of the invention may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Novel proteins having properties of interest may be createdby combining elements and fragments of proteins of the presentinvention, as well as with other proteins. Methods for suchmanipulations are generally known in the art. For example, amino acidsequence variants of the defensin proteins can be prepared by mutationsin the DNA. Methods for mutagenesis and nucleotide sequence alterationsare well known in the art. See, for example, Kunkel (1985) Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol.154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983)Techniques in Molecular Biology (Macmillan Publishing Company, New York)and the references cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al. (1978) Atlas ofProtein Sequence and Structure (Natl. Biomed. Res. Found., Washington,D.C.), herein incorporated by reference. Conservative substitutions,such as exchanging one amino acid with another having similarproperties, may be preferred.

Thus, the genes and nucleotide sequences of the invention include boththe naturally occurring sequences as well as mutant forms. Likewise, theproteins of the invention encompass naturally occurring proteins as wellas variations and modified forms thereof. Such variants will continue topossess the desired developmental activity, or defense responseactivity. Obviously, the mutations that will be made in the DNA encodingthe variant must not place the sequence out of reading frame andpreferably will not create complementary regions that could producesecondary mRNA structure. See, EP Patent No. 75,444.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays. That is, the activity can beevaluated by defensin activity assays. See, for example, Lancaster etal. (1994) J. Biol. Chem. 14:1137-1142 and Terras et al. (1995) PlantCell 7:537-588, herein incorporated by reference. Additionally,differences in the expression of specific genes between uninfected andinfected plants can be determined using gene expression profiling. RNAwas analyzed using the gene expression profiling process (GeneCalling®)as described in U.S. Pat. No. 5,871,697, herein incorporated byreference.

Variant nucleotide sequences and proteins also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more different defensincoding sequences can be manipulated to create a new defensin proteinpossessing the desired properties. In this manner, libraries ofrecombinant polynucleotides are generated from a population of relatedsequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. Strategies for such DNA shuffling are known in theart. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997)Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.Determination of preferred codons can be based on a survey of genesderived from the host cell where sequence information is available. Forexample, the codon frequency tables available on the World Wide Web atKazusa.or.jp/codon/may be used to determine preferred codons for avariety of organisms. See also Campbell and Gowri (1990) Plant Physiol.92:1-11; Murray et al. (1989) Nucleic Acids Res. 17:477-498, and U.S.Pat. Nos. 5,380,831 and 5,436,391; herein incorporated by reference.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments which may then beenzymatically assembled to construct the entire desired nucleic acidfragment. “Chemically synthesized”, as related to a nucleic acidfragment, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of nucleic acid fragments may be accomplishedusing well established procedures, or automated chemical synthesis canbe performed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of nucleotide sequence to reflect thecodon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

It is to be understood that as used herein the term “transgenic”includes any cell, cell line, callus, tissue, plant part, or plant thegenotype of which has been altered by the presence of a heterologousnucleic acid including those transgenics initially so altered as well asthose created by sexual crosses or asexual propogation from the initialtransgenic. The term “transgenic” as used herein does not encompass thealteration of the genome (chromosomal or extra-chromosomal) byconventional plant breeding methods or by naturally occurring eventssuch as random cross-fertilization, non-recombinant viral infection,non-recombinant bacterial transformation, non-recombinant transposition,or spontaneous mutation.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, andprogeny of same. Parts of transgenic plants are to be understood withinthe scope of the invention to comprise, for example, plant cells,protoplasts, tissues, callus, embryos as well as flowers, stems, fruits,leaves, roots originating in transgenic plants or their progenypreviously transformed with a DNA molecule of the invention andtherefore consisting at least in part of transgenic cells, are also anobject of the present invention.

As used herein, the term “plant cell” includes, without limitation,seeds suspension cultures, embryos, meristematic regions, callus tissue,leaves, roots, shoots, gametophytes, sporophytes, pollen, andmicrospores. The class of plants that can be used in the methods of theinvention is generally as broad as the class of higher plants amenableto transformation techniques, including both monocotyledonous anddicotyledonous plants.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. As used herein, the terms “encoding” or“encoded” when used in the context of a specified nucleic acid mean thatthe nucleic acid comprises the requisite information to guidetranslation of the nucleotide sequence into a specified protein. Theinformation by which a protein is encoded is specified by the use ofcodons. A nucleic acid encoding a protein may comprise non-translatedsequences (e.g., introns) within translated regions of the nucleic acidor may lack such intervening non-translated sequences (e.g., as incDNA).

“Regulatory sequences” refer to nucleotide sequences located upstream(5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters, translation leadersequences, introns, and polyadenylation recognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence that can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions.Promoters that cause a nucleic acid fragment to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”. New promoters of various types useful in plant cells areconstantly being discovered; numerous examples may be found in thecompilation by Okamuro and Goldberg (1989) Biochemistry of Plants15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

The “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be an RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptides by the cell. “cDNA” refers to a DNA that is complementaryto and derived from an mRNA template. The cDNA can be single-stranded orconverted to double stranded form using, for example, the Klenowfragment of DNA polymerase I. “Sense” RNA refers to an RNA transcriptthat includes the mRNA and so can be translated into a polypeptide bythe cell. “Antisense”, when used in the context of a particularnucleotide sequence, refers to the complementary strand of the referencetranscription product. “Antisense RNA” refers to an RNA transcript thatis complementary to all or part of a target primary transcript or mRNAand that blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous nucleotidesequence can be from a species different from that from which thenucleotide sequence was derived, or, if from the same species, thepromoter is not naturally found operably linked to the nucleotidesequence. A heterologous protein may originate from a foreign species,or, if from the same species, is substantially modified from itsoriginal form by deliberate human intervention.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Underexpression”refers to the production of a gene product in transgenic organisms atlevels below that of levels of production in normal or non-transformedorganisms. “Co-suppression” refers to the production of sense RNAtranscripts capable of suppressing the expression of identical orsubstantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020, incorporated herein by reference).

A “protein” or “polypeptide” is a chain of amino acids arranged in aspecific order determined by the coding sequence in a polynucleotideencoding the polypeptide. Each protein or polypeptide has a uniquefunction.

“Altered levels” or “altered expression” refers to the production ofgene product(s) in transgenic organisms in amounts or proportions thatdiffer from that of normal or non-transformed organisms.

“Null mutant” refers here to a host cell that either lacks theexpression of a certain polypeptide or expresses a polypeptide which isinactive or does not have any detectable expected enzymatic function.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product has been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be but are not limited tointracellular localization signals.

A “chloroplast transit peptide” is an amino acid sequence that istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence that is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal can furtherbe added, or if to the endoplasmic reticulum, an endoplasmic reticulumretention signal may be added. If the protein is to be directed to thenucleus, any signal peptide present should be removed and instead anuclear localization signal included (Raikhel (1992) Plant Phys.100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference). Additional transformation methods aredisclosed below. Thus, isolated polynucleotides of the present inventioncan be incorporated into recombinant constructs, typically DNAconstructs, capable of introduction into and replication in a host cell.Such a construct can be a vector that includes a replication system andsequences that are capable of transcription and translation of apolypeptide-encoding sequence in a given host cell. A number of vectorssuitable for stable transfection of plant cells or for the establishmentof transgenic plants have been described in, e.g., Pouwels et al.,(1985; Supp. 1987) Cloning Vectors: A Laboratory Manual, Weissbach andWeissbach (1989) Methods for Plant Molecular Biology, (Academic Press,New York); and Flevin et al., (1990) Plant Molecular Biology Manual,(Kluwer Academic Publishers). Typically, plant expression vectorsinclude, for example, one or more cloned plant genes under thetranscriptional control of 5′ and 3′ regulatory sequences and a dominantselectable marker. Such plant expression vectors also can contain apromoter regulatory region (e.g., a regulatory region controllinginducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific expression), atranscription initiation start site, a ribosome binding site, an RNAprocessing signal, a transcription termination site, and/or apolyadenylation signal.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual; (2d ed.; Cold SpringHarbor Laboratory Press) Plainview, N.Y., hereinafter referred to as“Maniatis”.

“PCR” or “polymerase chain reaction” is a technique used for theamplification of specific DNA segments (U.S. Pat. Nos. 4,683,195 and4,800,159).

The present invention concerns an isolated polynucleotide comprising anucleotide sequence selected from the group consisting of: a nucleotidesequence set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 46, or 48; a nucleotide sequence that encodes apolypeptide having the amino acid sequence set forth in SEQ ID NO: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 32, 47, or 49; anucleotide sequence that encodes a mature polypeptide having the aminoacid sequence set forth in SEQ ID NO: 35; a nucleotide sequencecharacterized by at least 75% sequence identity to the nucleotidesequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 46, or 48; a nucleotide sequence characterized by atleast 80% sequence identity to the nucleotide sequence set forth in SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 46, or48; a nucleotide sequence characterized by at least 85% sequenceidentity to the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 46, or 48; a nucleotidesequence characterized by at least 90% sequence identity to thenucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 46, or 48; and a nucleotide sequencethat comprises the complement of any one of the above.

Nucleic acid fragments encoding at least a portion of several plantdefensins have been isolated and identified by comparison of randomplant cDNA sequences to public databases containing nucleotide andprotein sequences using the BLAST algorithms well known to those skilledin the art. The nucleic acid fragments of the instant invention may beused to isolate cDNAs and genes encoding homologous proteins from thesame or other plant species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction, ligase chain reaction).

For example, genes encoding other plant defensins, either as cDNAs orgenomic DNAs, could be isolated directly by using all or a portion ofthe instant nucleic acid fragments as DNA hybridization probes to screenlibraries from any desired plant employing methodology well known tothose skilled in the art. Specific oligonucleotide probes based upon theinstant nucleic acid sequences can be designed and synthesized bymethods known in the art (Maniatis). Moreover, the entire sequences canbe used directly to synthesize DNA probes by methods known to theskilled artisan such as random primer DNA labeling, nick translation, orend-labeling techniques, or RNA probes using available in vitrotranscription systems. In addition, specific primers can be designed andused to amplify a part or all of the instant sequences. The resultingamplification products can be labeled directly during amplificationreactions or labeled after amplification reactions, and used as probesto isolate full length cDNA or genomic fragments under conditions ofappropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002)to generate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl.Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220).Products generated by the 3′ and 5′ RACE procedures can be combined togenerate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165). Consequently, a polynucleotide comprising a nucleotide sequence ofat least one of 60 (preferably one of at least 40, most preferably oneof at least 30) contiguous nucleotides derived from a nucleotidesequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 46, or 48, and thecomplement of such nucleotide sequences may be used in such methods toobtain a nucleic acid fragment encoding a substantial portion of anamino acid sequence of a polypeptide.

The present invention relates to a method of obtaining a nucleic acidfragment encoding a substantial portion of a defensin polypeptidecomprising the steps of: synthesizing an oligonucleotide primercomprising a nucleotide sequence of at least one of 60 (preferably atleast one of 40, most preferably at least one of 30) contiguousnucleotides derived from a nucleotide sequence selected from the orconsisting of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 46, or 48, and the complement of such nucleotide sequences;and amplifying a nucleic acid fragment (preferably a cDNA inserted in acloning vector) using the oligonucleotide primer. The amplified nucleicacid fragment preferably will encode a portion of a plant defensinpolypeptide.

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol.36:1-34; Maniatis).

In another embodiment, this invention concerns viruses and host cellscomprising either the chimeric genes of the invention as describedherein or an isolated polynucleotide of the invention as describedherein. Examples of host cells that can be used to practice theinvention include, but are not limited to, yeast, bacteria, fungus,insect, mammalian, and plant cells.

By “host cell” a cell, which comprises a heterologous nucleic acidsequence of the invention is meant. Host cells may be prokaryotic cellssuch as E. coli, or eukaryotic cells such as yeast, insect, amphibian,or mammalian cells. Preferably, host cells are monocotyledonous ordicotyledonous plant cells. A particularly preferred monocotyledonoushost cell is a maize host cell.

Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.The chimeric gene may comprise promoter sequences and translation leadersequences derived from the same genes. 3′ non-coding sequences encodingtranscription termination signals may also be provided. The instantchimeric gene may also comprise one or more introns in order tofacilitate gene expression.

The defensin sequences of the invention are provided in expressioncassettes or DNA constructs for expression in the plant of interest. Thecassette will include 5′ and 3′ regulatory sequences operably linked toa defensin sequence of the invention. The cassette may additionallycontain at least one additional gene to be cotransformed into theorganism. Alternatively, the additional gene(s) can be provided onmultiple expression cassettes.

Such an expression cassette is provided with a plurality of restrictionsites for insertion of the defensin sequence to be under thetranscriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, adefensin DNA sequence of the invention, and a transcriptional andtranslational termination region functional in plants. Thetranscriptional initiation region, the promoter, may be native oranalogous or foreign or heterologous to the plant host. Additionally,the promoter may be the natural sequence or alternatively a syntheticsequence. By “foreign” is intended that the transcriptional initiationregion is not found in the native plant into which the transcriptionalinitiation region is introduced. As used herein, a chimeric genecomprises a coding sequence operably linked to a transcriptioninitiation region that is heterologous to the coding sequence.

While it may be preferable to express the sequences using heterologouspromoters, the native promoter sequences may be used. Such constructswould change expression levels of defensin in the host cell (i.e., plantor plant cell). Thus, the phenotype of the host cell (i.e., plant orplant cell) is altered.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,or may be derived from another source. Convenient termination regionsare available from the Ti-plasmid of A. tumefaciens, such as theoctopine synthase and nopaline synthase (NOS) termination regions. Seealso Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; andJoshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5′ leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translation leaders are known in the art andinclude: picomavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238); MDMVleader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and humanimmunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991)Nature 353:90-94); untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) inMolecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); andmaize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991)Virology 81:382-385). See also, Della-Cioppa et al. (1987) PlantPhysiol. 84:965-968. Other methods known to enhance translation can alsobe utilized, for example, introns, and the like.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues. Marker genesinclude genes encoding antibiotic resistance, such as those encodingneomycin phosphotransferase II (NEO) and hygromycin phosphotransferase(HPT), as well as genes conferring resistance to herbicidal compounds,such as glyphosate, glufosinate ammonium, bromoxynil, imidazolinones,and 2,4-dichlorophenoxyacetate (2,4-D). See generally, Yarranton (1992)Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl.Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff(1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon,pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989)Proc. Natl. Acad. Sci. USA 86:5400-5404; Fuerst et al. (1989) Proc.Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg;Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow etal. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) Proc.Natl. Acad. Sci. USA 89:3952-3956; Baim et al. (1991) Proc. Natl. Acad.Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res.19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol.10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother.35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104;Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al.(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992)Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbookof Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill etal. (1988) Nature 334:721-724. Such disclosures are herein incorporatedby reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the present invention.

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. That is, thenucleic acids can be combined with constitutive, tissue-preferred, orother promoters for expression in the host cell of interest. Suchconstitutive promoters include, for example, the core promoter of theRsyn7 promoter and other constitutive promoters disclosed in WO 99/43838and U.S. Pat. No. 6,072,050; the core CaMV ³⁵S promoter (Odell et al.(1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol.12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689);pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten etal. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026),and the like. Other constitutive promoters include, for example, thosedisclosed in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611, hereinincorporated by reference.

Generally, it will be beneficial to express the gene from an induciblepromoter, particularly from a pathogen-inducible promoter. Suchpromoters include those from pathogenesis-related proteins (PRproteins), which are induced following infection by a pathogen; e.g., PRproteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, forexample, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Ukneset al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol.Virol. 4:111-116. See also WO 99/43819 published Sep. 9, 1999, hereinincorporated by reference.

Of interest are promoters that are expressed locally at or near the siteof pathogen infection. See, for example, Marineau et al. (1987) PlantMol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; andYang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen etal. (1996) Plant J 10:955-966; Zhang et al. (1994) Proc. Natl. Acad.Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertzet al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible); and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200).

Additionally, as pathogens find entry into plants through wounds orinsect damage, a wound-inducible promoter may be used in theconstructions of the invention. Such wound-inducible promoters includepotato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev.Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2(Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurlet al. (1992) Science 225:1570-1573); WIPI (Rohmeier et al. (1993) PlantMol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76);MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like,herein incorporated by reference.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced defensinexpression within a particular plant tissue. Tissue-preferred promotersinclude Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al.(1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. GenGenet. 254(3):337-343; Russell et al. (1997) Transgenic Res.6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341;Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al.(1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant CellPhysiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ.20:181-196; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138;Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; andGuevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters canbe modified, if necessary, for weak expression.

Leaf-specific promoters are known in the art. See, for example, Yamamotoet al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol.105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778;Gotor et al. (1993) Plant J 3:509-18; Orozco et al. (1993) Plant Mol.Biol. 23(6):1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci.USA 90(20):9586-9590.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphatesynthase); and celA (cellulose synthase) (see WO 00/11177, hereinincorporated by reference). Gama-zein is a preferred endosperm-specificpromoter. Glob-1 is a preferred embryo-specific promoter. For dicots,seed-specific promoters include, but are not limited to, beanβ-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and thelike. For monocots, seed-specific promoters include, but are not limitedto, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken1, shrunken 2, globulin 1, etc. See also WO 00/12733, whereseed-preferred promoters from end1 and end2 genes are disclosed; hereinincorporated by reference.

The method of transformation/transfection is not critical to the instantinvention. Various methods of transformation or transfection arecurrently available. As newer methods are available to transform cropsor other host cells they may be used with the instant invention.Accordingly, a wide variety of methods have been developed to insert aDNA sequence into the genome of a host cell to obtain the transcriptionand/or translation of the sequence to effect phenotypic changes in theorganism. The nucleic acid fragments of the instant invention may beused to create transgenic plants in which the disclosed plant defensinare present at higher or lower levels than normal or in cell types ordevelopmental stages in which they are not normally found. This wouldhave the effect of altering the level of disease (e.g., fungal)resistance and stress tolerance in those cells. Thus, any method, whichprovides for effective transformation/transfection may be employed.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606,Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No.5,563,055; Zhao et al., U.S. Pat. No. 5,981,840), direct gene transfer(Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particleacceleration (see, for example, Sanford et al., U.S. Pat. No. 4,945,050;Tomes et al., U.S. Pat. No. 5,879,918; Tomes et al., U.S. Pat. No.5,886,244; Bidney et al., U.S. Pat. No. 5,932,782; McCabe et al. (1988)Biotechnology 6:923-926); and Lecl transformation (WO 00/28058). Alsosee Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al.(1987) Particulate Science and Technology 5:27-37 (onion); Christou etal. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl.Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes,U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and5,324,646; Tomes et al. (1995) “Direct DNA Transfer into Intact PlantCells via Microprojectile Bombardment,” in Plant Cell, Tissue, and OrganCulture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin)(maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm etal. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren etal. (1984) Nature (London) 311:763-764; Bowen et al., U.S. Pat. No.5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensurethat expression of the desired phenotypic characteristic has beenachieved.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplants of interest include, but are not limited to, corn (Zea mays),Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly thoseBrassica species useful as sources of seed oil, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatas), cassaya (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadarnia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp., Pisum spp.), and members of the genusCucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis),and musk melon (C. melo). Ornamentals include azalea (Rhododendronspp.), hydrangea (Hydrangea macrophylla), hibiscus (Hibiscusrosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils(Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthuscaryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). Preferably, plants of the presentinvention are crop plants (for example, corn, alfalfa, sunflower,Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet,tobacco, etc.), more preferably corn and soybean plants, yet morepreferably corn plants.

Prokaryotic cells may be used as hosts for expression. Prokaryotes mostfrequently are represented by various strains of E. coli; however, othermicrobial strains may also be used. Commonly used prokaryotic controlsequences which are defined herein to include promoters fortranscription initiation, optionally with an operator, along withribosome binding sequences, include such commonly used promoters as thebeta lactamase (penicillinase) and lactose (lac) promoter systems (Changet al. (1977) Nature 198:1056), the tryptophan (trp) promoter system(Goeddel et al. (1980) Nucleic Acids Res. 8:4057) and the lambda derivedPL promoter and N-gene ribosome binding site (Simatake and Rosenberg(1981) Nature 292:128). Examples of selection markers for E. coliinclude, for example, genes specifying resistance to ampicillin,tetracycline, or chloramphenicol.

The vector is selected to allow introduction into the appropriate hostcell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for expressing a protein of the present invention areavailable using Bacillus sp. and Salmonella (Palva et al. (1983) Gene22:229-235 and Mosbach et al. (1983) Nature 302:543-545).

A variety of eukaryotic expression systems such as yeast, insect celllines, plant and mammalian cells, are known to those of skill in theart. As explained briefly below, a polynucleotide of the presentinvention can be expressed in these eukaryotic systems. In someembodiments, transformed/transfected plant cells, as discussed infra,are employed as expression systems for production of the proteins of theinstant invention. Such antimicrobial proteins can be used for anyapplication including coating surfaces to target microbes as describedfurther infra.

Synthesis of heterologous nucleotide sequences in yeast is well known.Sherman, et al. (1982) Methods in Yeast Genetics (Cold Spring HarborLaboratory) is a well recognized work describing the various methodsavailable to produce proteins in yeast. Two widely utilized yeasts forproduction of eukaryotic proteins are Saccharomyces cerevisiae andPichia pastoris. Vectors, strains, and protocols for expression inSaccharomyces and Pichia are known in the art and available fromcommercial suppliers (e.g., Invitrogen). Suitable vectors usually haveexpression control sequences, such as promoters, including3-phosphoglycerate kinase or alcohol oxidase, and an origin ofreplication, termination sequences and the like, as desired.

A protein of the present invention, once expressed, can be isolated fromyeast by lysing the cells and applying standard protein isolationtechniques to the lysates. The monitoring of the purification processcan be accomplished by using Western blot techniques, radioimmunoassay,or other standard immunoassay techniques.

The sequences of the present invention can also be ligated to variousexpression vectors for use in transfecting cell cultures of, forinstance, mammalian, insect, or plant origin. Illustrative cell culturesuseful for the production of the peptides are mammalian cells. A numberof suitable host cell lines capable of expressing intact proteins havebeen developed in the art, and include the HEK293, BHK21, and CHO celllines. Expression vectors for these cells can include expression controlsequences, such as an origin of replication, a promoter (e.g. the CMVpromoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter),an enhancer (Queen et al. (1986) Immunol. Rev. 89:49), and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites (e.g., an SV40 large T Ag poly A additionsite), and transcriptional terminator sequences. Other animal cellsuseful for production of proteins of the present invention areavailable, for instance, from the American Type Culture Collection.

Appropriate vectors for expressing proteins of the present invention ininsect cells are usually derived from the SF9 baculovirus. Suitableinsect cell lines include mosquito larvae, silkworm, armyworm, moth andDrosophila cell lines such as a Schneider cell line (See, Schneider(1987) J. Embryol. Exp. Morphol. 27:353-365).

As with yeast, when higher animal or plant host cells are employed,polyadenylation or transcription terminator sequences are typicallyincorporated into the vector. An example of a terminator sequence is thepolyadenylation sequence from the bovine growth hormone gene. Sequencesfor accurate splicing of the transcript may also be included. An exampleof a splicing sequence is the VP1 intron from SV40 (Sprague et al.(1983) J. Virol. 45:773-781). Additionally, gene sequences to controlreplication in the host cell may be incorporated into the vector such asthose found in bovine papilloma virus type-vectors. Saveria-Campo (1985)“Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector,” in DNA CloningVol. II. A Practical Approach, ed. D. M. Glover (IRL Press, Arlington,Va.), pp. 213-238.

Animal and lower eukaryotic (e.g., yeast) host cells are competent orrendered competent for transfection by various means. There are severalwell-known methods of introducing DNA into animal cells. These include:calcium phosphate precipitation, fusion of the recipient cells withbacterial protoplasts containing the DNA, treatment of the recipientcells with liposomes containing the DNA, DEAE dextrin, electroporation,biolistics, and micro-injection of the DNA directly into the cells. Thetransfected cells are cultured by means well known in the art. Kuchler(1997) Biochemical Methods in Cell Culture and Virology (Dowden,Hutchinson and Ross, Inc.).

Plasmid vectors comprising the instant isolated polynucleotide (orchimeric gene) may be constructed. The choice of plasmid vector isdependent upon the method that will be used to transform host plants.The skilled artisan is well aware of the genetic elements that must bepresent on the plasmid vector in order to successfully transform, selectand propagate host cells containing the chimeric gene. The skilledartisan will also recognize that different independent transformationevents will result in different levels and patterns of expression (Joneset al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen.Genetics 218:78-86), and thus that multiple events must be screened inorder to obtain lines displaying the desired expression level andpattern. Such screening may be accomplished by Southern analysis of DNA,Northern analysis of mRNA expression, Western analysis of proteinexpression, or phenotypic analysis.

For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by directing the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100: 1627-1632) with or without removingtargeting sequences that are already present. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of use may be discovered in the future.

It may also be desirable to reduce or eliminate expression of genesencoding the instant polypeptides in plants for some applications. Inorder to accomplish this, a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

Molecular genetic solutions to the generation of plants with alteredgene expression have a decided advantage over more traditional plantbreeding approaches. Changes in plant phenotypes can be produced byspecifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression of aspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations areassociated with the use of antisense or cosuppression technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of sense or antisense genes may require the use ofdifferent chimeric genes utilizing different regulatory elements knownto the skilled artisan. Once transgenic plants are obtained by one ofthe methods described above, it will be necessary to screen individualtransgenics for those that most effectively display the desiredphenotype. Accordingly, the skilled artisan will develop methods forscreening large numbers of transformants. The nature of these screenswill generally be chosen on practical grounds. For example, one canscreen by looking for changes in gene expression by using antibodiesspecific for the protein encoded by the gene being suppressed, or onecould establish assays that specifically measure enzyme activity. Apreferred method will be one that allows large numbers of samples to beprocessed rapidly, since it will be expected that a large number oftransformants will be negative for the desired phenotype.

The present invention also provides an isolated polypeptide selectedfrom the group consisting of: a polypeptide comprising an amino acidsequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 32, 47, or 49; a polypeptide characterized by at least 80%identity to SEQ ID NO: 6, 26, or 28; a polypeptide characterized by atleast 85% identity to SEQ ID NO: 8; a polypeptide characterized by atleast 95% identity to SEQ ID NO: 2, 4, 10, 12, 14, 16, 18, 20, 22, 24,or 47; a polypeptide characterized by at least 97% identity to SEQ IDNO: 32; and the polypeptide of SEQ ID NO: 49.

The instant polypeptides are useful in methods for impacting a plantpathogen comprising introducing into a plant or cell thereof at leastone nucleotide construct comprising a nucleotide sequence of theinvention operably linked to a promoter that drives expression of anoperably linked sequence in plant cells, wherein said nucleotidesequence is selected from the group consisting of: a nucleotide sequenceset forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 46, or 48; a nucleotide sequence that encodes a polypeptidehaving the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 32, 47, or 49; a nucleotide sequencethat encodes a mature polypeptide having the amino acid sequence setforth in SEQ ID NO: 35; a nucleotide sequence characterized by at least75% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 46, or 48; anucleotide sequence characterized by at least 80% sequence identity tothe nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 46, or 48; a nucleotide sequencecharacterized by at least 85% sequence identity to the nucleotidesequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 46, or 48; a nucleotide sequence characterized by atleast 90% sequence identity to the nucleotide sequence set forth in SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 46, or48; and a nucleotide sequence that comprises the complement of any oneof the above.

The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to the these proteins by methodswell known to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Polyclonal defensin-like antibodies can beprepared by immunizing a suitable subject (e.g., rabbit, goat, mouse, orother mammal) with an defensive agent immunogen. The anti-defensinantibody titer in the immunized subject can be monitored over time bystandard techniques, such as with an enzyme linked immunosorbent assay(ELISA) using immobilized antimicrobial polypeptides. At an appropriatetime after immunization, e.g., when the anti-defensive agent antibodytiters are highest, antibody-producing cells can be obtained from thesubject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975) Nature 256:495-497, the human B cellhybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72), theEBV-hybridoma technique (Cole et al. (1985) in Monoclonal Antibodies andCancer Therapy, ed. Reisfeld and Sell (Alan R. Liss, Inc., New York,N.Y.), pp. 77-96) or trioma techniques. The technology for producinghybridomas is well known (see generally Coligan et al., eds. (1994)Current Protocols in Immunology (John Wiley & Sons, Inc., New York,N.Y.); Galfre et al. (1977) Nature 266:550-52; Kenneth (1980) inMonoclonal Antibodies: A New Dimension In Biological Analyses (PlenumPublishing Corp., New York); and Lerner (1981) Yale J. Biol. Med.54:387-402).

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-defensin-like antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with a defensin to thereby isolateimmunoglobulin library members that bind the defensive agent. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening an antibodydisplay library can be found in, for example, U.S. Pat. No. 5,223,409;PCT Publication Nos. WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679;93/01288; WO 92/01047; 92/09690; and 90/02809; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J. 12:725-734. The antibodies can be used to identifyhomologs of the defensins of the invention.

All or a substantial portion of the polynucleotides of the instantinvention may also be used as probes for genetically and physicallymapping the genes that they are a part of, and as markers for traitslinked to those genes. Such information may be useful in plant breedingin order to develop lines with desired phenotypes. For example, theinstant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4:37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al. in: Nonmammalian Genomic Analysis: APractical Guide, Academic Press, New York), 1996, pp. 319-346, andreferences cited therein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequences may be used in direct fluorescence in situhybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).Although current methods of FISH mapping favor use of large clones(several to several hundred KB; see Laan et al. (1995) Genome Res.5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat.Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic AcidRes. 17:6795-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instantcDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995)Proc. Natl. Acad. Sci. USA 92:8149-8153; Bensen et al. (1995) Plant Cell7:75-84). The latter approach may be accomplished in two ways. First,short segments of the instant nucleic acid fragments may be used inpolymerase chain reaction protocols in conjunction with a mutation tagsequence primer on DNAs prepared from a population of plants in whichMutator transposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptide.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptide can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptides disclosed herein.

Compositions and methods for controlling pathogenic agents are providedin the present invention. The anti-pathogenic compositions compriseplant defensin nucleotide and amino acid sequences. Particularly, theplant nucleic acid and amino acid sequences and fragments and variantsthereof set forth herein possess anti-pathogenic activity. Accordingly,the compositions and methods are useful in protecting plants againstfungal pathogens, viruses, nematodes, insects, and the like.Additionally provided are transformed plants, plant cells, plant tissuesand seeds thereof.

By “plant pathogen” or “plant pest” any organism that can cause harm toa plant, by inhibiting or slowing the growth of a plant, by damaging thetissues of a plant, by weakening the immune system of a plant, reducingthe resistance of a plant to abiotic stresses, and/or by causing thepremature death of the plant, etc., is intended. Plant pathogens andplant pests include insects, nematodes, and organisms such as fungi,viruses, and bacteria.

By “disease resistance” or “pathogen resistance” it is intended that theorganisms avoid the disease symptoms that are the outcome oforganism-pathogen interactions. That is, pathogens are prevented fromcausing diseases and the associated disease symptoms, or alternatively,the disease symptoms caused by the pathogen is minimized or lessened.

By “anti-pathogenic compositions” it is intended that the compositionsof the invention are capable of suppressing, controlling, and/or killingthe invading pathogenic organism. An antipathogenic composition of theinvention will reduce the disease symptoms resulting from pathogenchallenge by at least about 5% to about 50%, at least about 10% to about60%, at least about 30% to about 70%, at least about 40% to about 80%,or at least about 50% to about 90% or greater. Hence, the methods of theinvention can be utilized to protect plants from disease, particularlythose diseases that are caused by plant pathogens.

An “antimicrobial agent,” a “pesticidal agent,” a “defensin,” an“antiviral agent,” an “insecticidal agent,” and/or a “fugicidal agent”will act similarly to suppress, control, and/or kill the invadingpathogen.

A defensive agent will possess defensive activity. By “defensiveactivity” an antipathogenic, antimicrobial, antiviral, insecticidal, orantifungal activity is intended.

By “antipathogenic compositions” it is intended that the compositions ofthe invention have activity against pathogens; including fungi,microorganisms, viruses, insects, and nematodes, and thus are capable ofsuppressing, controlling, and/or killing the invading pathogenicorganism. An antipathogenic composition of the invention will reduce thedisease symptoms resulting from pathogen challenge by at least about 5%to about 50%, at least about 10% to about 60%, at least about 30% toabout 70%, at least about 40% to about 80%, or at least about 50% toabout 90% or greater. Hence, the methods of the invention can beutilized to protect organisms, particularly plants, from disease,particularly those diseases that are caused by invading pathogens.

Assays that measure antipathogenic activity are commonly known in theart, as are methods to quantitate disease resistance in plants followingpathogen infection. See, for example, U.S. Pat. No. 5,614,395, hereinincorporated by reference. Such techniques include, measuring over time,the average lesion diameter, the pathogen biomass, and the overallpercentage of decayed plant tissues. For example, a plant eitherexpressing an antipathogenic polypeptide or having an antipathogeniccomposition applied to its surface shows a decrease in tissue necrosis(i.e., lesion diameter) or a decrease in plant death following pathogenchallenge when compared to a control plant that was not exposed to theantipathogenic composition. Alternatively, antipathogenic activity canbe measured by a decrease in pathogen biomass. For example, a plantexpressing an antipathogenic polypeptide or exposed to an antipathogeniccomposition is challenged with a pathogen of interest. Over time, tissuesamples from the pathogen-inoculated tissues are obtained and RNA isextracted. The percent of a specific pathogen RNA transcript relative tothe level of a plant specific transcript allows the level of pathogenbiomass to be determined. See, for example, Thomma et al. (1998) PlantBiology 95:15107-15111, herein incorporated by reference.

Furthermore, in vitro antipathogenic assays include, for example, theaddition of varying concentrations of the antipathogenic composition topaper disks and placing the disks on agar containing a suspension of thepathogen of interest. Following incubation, clear inhibition zonesdevelop around the discs that contain an effective concentration of theantipathogenic polypeptide (Liu et al. (1994) Plant Biology91:1888-1892, herein incorporated by reference). Additionally,microspectrophotometrical analysis can be used to measure the in vitroantipathogenic properties of a composition (Hu et al. (1997) Plant Mol.Biol. 34:949-959 and Cammue et al. (1992) J. Biol. Chem. 267: 2228-2233,both of which are herein incorporated by reference).

In specific embodiments, methods for increasing pathogen resistance in aplant comprise stably transforming a plant with a DNA constructcomprising an antipathogenic nucleotide sequence of the inventionoperably linked to a promoter that drives expression in a plant. Suchmethods find use in agriculture particularly in limiting the impact ofplant pathogens on crop plants. While the choice of promoter will dependon the desired timing and location of expression of the anti-pathogenicnucleotide sequences, preferred promoters include constitutive andpathogen-inducible promoters.

It is understood in the art that plant DNA viruses and fungal pathogensremodel the control of the host replication and gene expressionmachinery to accomplish their own replication and effective infection.The present invention may be useful in preventing such corruption of thecell.

The defensin sequences find use in disrupting cellular function of plantpathogens or insect pests as well as altering the defense mechanisms ofa host plant to enhance resistance to disease or insect pests. While theinvention is not bound by any particular mechanism of action to enhancedisease resistance, the gene products of the defensin sequences functionto inhibit or prevent diseases in a plant.

The methods of the invention can be used with other methods available inthe art for enhancing disease resistance in plants. For example, any oneof a variety of second nucleotide sequences may be utilized, embodimentsof the invention encompass those second nucleotide sequences that, whenexpressed in a plant, help to increase the resistance of a plant topathogens. It is recognized that such second nucleotide sequences may beused in either the sense or antisense orientation depending on thedesired outcome. Other plant defense proteins include those described inPCT patent publications WO 99/43823 and WO 99/43821, both of which areherein incorporated by reference.

Pathogens of the invention include, but are not limited to, viruses orviroids, bacteria, insects, nematodes, fingi, and the like. Virusesinclude any plant virus, for example, tobacco or cucumber mosaic virus,ringspot virus, necrosis virus, maize dwarf mosaic virus, etc. Specificfungal and viral pathogens for the major crops include: Soybeans:Phytophthora megasperma f.sp. glycinea, Macrophomina phaseolina,Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum,Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthephaseolorum var. caulivora, Sclerotium rolfsii, Cercospora kikuchii,Cercospora sojina, Peronospora manshurica, Colletotrichum dematium(Colletotrichum truncatum), Corynespora cassiicola, Septoria glycines,Phyllosticta sojicola, Alternaria alternata, Pseudomonas syringae p.v.glycinea, Xanthomonas campestris p.v. phaseoli, Microsphaera diffusa,Fusarium semitectum, Phialophora gregata, Soybean mosaic virus,Glomerella glycines, Tobacco Ring spot virus, Tobacco Streak virus,Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythiumdebaryanum, Tomato spotted wilt virus, Heterodera glycines, Fusariumsolani; Canola: Albugo candida, Alternaria brassicae, Leptosphaeriamaculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerellabrassiccola, Pythium ultimum, Peronospora parasitica, Fusarium roseum,Alternaria alternata; Alfalfa: Clavibacter Michigan's subsp. insidiosum,Pythium ultimum, Pythium irregulare, Pythium splendens, Pythiumdebaryanum, Pythium aphanidermatum, Phytophthora megasperma, Peronosporatrifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis,Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium spp.,Xanthomonas campestris p.v. alfalfae, Aphanomyces euteiches, Stemphyliumherbarum, Stemphylium alfalfae; Wheat: Pseudomonas syringae p.v.atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v.translucens, Pseudomonas syringae p.v. syringae, Alternaria alternata,Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum,Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporiumgramineum, Collotetrichum graminicola, Erysiphe graminis f.sp. tritici,Puccinia graminis f.sp. tritici, Puccinia recondita f.sp. tritici,Puccinia striiformis, Pyrenophora tritici-repentis, Septoria nodorum,Septoria tritici, Septoria avenae, Pseudocercosporella herpotrichoides,Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var.tritici, Pythium aphanidermatum, Pythium arrhenomanes, Pythium ultimum,Bipolaris sorokiniana, Barley Yellow Dwarf Virus, Brome Mosaic Virus,Soil Borne Wheat Mosaic Virus, Wheat Streak Mosaic Virus, Wheat SpindleStreak Virus, American Wheat Striate Virus, Claviceps purpurea, Tilletiatritici, Tilletia laevis, Tilletia indica, Pythium gramicola, HighPlains Virus, European wheat striate virus; Sunflower: Plasmophorahalstedii, Sclerotinia sclerotiorum, Aster Yellows, Septoria helianthi,Phomopsis helianthi, Alternaria helianthi, Alternaria zinniae, Botrytiscinerea, Phoma macdonaldii, Macrophomina phaseolina, Erysiphecichoracearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer,Puccinia helianthi, Verticillium dahliae, Erwinia carotovorum p.v.carotovora, Cephalosporium acremonium, Phytophthora cryptogea, Albugotragopogonis; Corn: Fusarium moniliforme var. subglutinans, Erwiniastewartii, Fusarium moniliforme, Gibberella zeae (Fusarium graminearum),Stenocarpella maydis (Diplodia maydis), Pythium irregulare, Pythiumdebaryanum, Pythium graminicola, Pythium splendens, Pythium ultimum,Pythium aphanidermatum, Aspergillus flavus, Bipolaris maydis O, T(Cochliobolus heterostrophus), Helminthosporium carbonum I, II & III(Cochliobolus carbonum), Exserohilum turcicum I, II & III,Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis,Kabatiella maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi,Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum,Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvulariainaequalis, Curvularia pallescens, Clavibacter michiganense subsp.nebraskense, Trichoderma viride, Maize Dwarf Mosaic Virus A & B, WheatStreak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi,Pseudomonas avenae, Erwinia chrysanthemi p.v. zea, Erwinia carotovora,Corn stunt spiroplasma, Diplodia macrospora, Sclerophthora macrospora,Peronosclerospora sorghi, Peronosclerospora philippinensis,Peronosclerospora maydis, Peronosclerospora sacchari, Sphacelothecareiliana, Physopella zeae, Cephalosporium maydis, Cephalosporiumacremonium, Maize Chlorotic Mottle Virus, High Plains Virus, MaizeMosaic Virus, Maize Rayado Fino Virus, Maize Streak Virus, Maize StripeVirus, Maize Rough Dwarf Virus; Sorghum: Exserohilum turcicum,Colletotrichum graminicola (Glomerella graminicola), Cercospora sorghi,Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae p.v.syringae, Xanthomonas campestris p.v. holcicola, Pseudomonasandropogonis, Puccinia purpurea, Macrophomina phaseolina, Periconiacircinata, Fusarium moniliforme, Alternaria alternata, Bipolarissorghicola, Helminthosporium sorghicola, Curvularia lunata, Phomainsidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans), Ramulisporasorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisoriumreilianum (Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisoriumsorghi, Sugarcane mosaic H, Maize Dwarf Mosaic Virus A & B, Clavicepssorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthonamacrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis,Sclerospora graminicola, Fusarium graminearum, Fusarium oxysporum,Pythium arrhenomanes, Pythium graminicola, etc.

Nematodes include parasitic nematodes such as root-knot, cyst, andlesion nematodes, including Heterodera and Globodera spp.; particularlyGlobodera rostochiensis and Globodera pailida (potato cyst nematodes);Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beetcyst nematode); and Heterodera avenae (cereal cyst nematode). Additionalnematodes include: Heterodera cajani; Heterodera trifolii; Heteroderaoryzae; Globodera tabacum; Meloidogyne incognita; Meloidogynejavonica;Meloidogynehapla; Meloidogyne arenaria; Meloidogynenaasi;Meloidogyneexigua; Xiphinema index; Xiphinema italiae; Xiphinemaamericanum; Xiphinema diversicaudatum; Pratylenchus penetrans;Pratylenchus brachyurus; Pratylenchus zeae; Pratylenchus coffeae;Pratylenchus thornei; Pratylenchus scribneri; Pratylenchus vulnus;Pratylenchus curvitatus; Radopholus similis; Radopholus citrophilus;Ditylenchus dipsaci; Helicotylenchus multicintus; Rotylenchulusreniformis; Belonolaimus spp.; Paratrichodorus anemones; Trichodorusspp.; Primitivus spp.; Anguina tritici; Bider avenae; Subanguinaradicicola; Tylenchorhynchus spp.; Haplolaimus seinhorsti; Tylenchulussemipenetrans; Hemicycliophora arenaria; Belonolaimus langicaudatus;Paratrichodorus xiphinema; Paratrichodorus christiei; Rhadinaphelenchuscocophilus; Paratrichodorus minor; Hoplolaimus galeatus; Hoplolaimuscolumbus; Criconemella spp.; Paratylenchus spp.; Nacoabbus aberrans;Aphelenchoides besseyi; Ditylenchus angustus; Hirchmaniella spp.;Scutellonema spp.; Hemicriconemoides kanayaensis; Tylenchorynchusclaytoni; and Cacopaurus pestis.

Insect pests include insects selected from the orders Coleoptera,Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera,Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera,Trichoptera, etc., particularly Coleoptera and Lepidoptera. Insect pestsof the invention for the major crops include: Maize: Ostrinia nubilalis,European corn borer; Agrotis ipsilon, black cutworm; Helicoverpa zea,corn earworm; Spodoptera frugiperda, fall armyworm; Diatraeagrandiosella, southwestern corn borer; Elasmopalpus lignosellus, lessercornstalk borer; Diatraea saccharalis, sugarcane borer; Diabroticavirgifera, western corn rootworm; Diabrotica longicornis barberi,northern corn rootworm; Diabrotica undecimpunctata howardi, southerncorn rootworm; Melanotus spp., wireworms; Cyclocephala borealis,northern masked chafer (white grub); Cyclocephala immaculata, southernmasked chafer (white grub); Popillia japonica, Japanese beetle;Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maizebillbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis,corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplusfemurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratorygrasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis,corn blotch leafminer; Anaphothrips obscrurus, grass thrips; Solenopsismilesta, thief ant; Tetranychus urticae, twospotted spider mite;Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fallarmyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus,lesser cornstalk borer; Feltia subterranea, granulate cutworm;Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp.,wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria,corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphummaidis; corn leaf aphid; Sipha flava, yellow sugarcane aphid; Blissusleucopterus leucopterus, chinch bug; Contarinia sorghicola, sorghummidge; Tetranychus cinnabarinus, carmine spider mite; Tetranychusurticae, twospotted spider mite; Wheat: Pseudaletia unipunctata, armyworm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus,lesser cornstalk borer; Agrotis orthogonia, western cutworm;Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus,cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabroticaundecimpunctata howardi, southern corn rootworm; Russian wheat aphid;Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid;Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Melanoplus sanguinipes,migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosismosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemyacoarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephuscinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower:Suleima helianthana, sunflower bud moth; Homoeosoma electellum,sunflower moth; Zygogramma exclamationis, sunflower beetle; Bothyrusgibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seedmidge; Cotton: Heliothis virescens, cotton budworm; Helicoverpa zea,cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophoragossypiella, pink bollworm; Anthonomus grandis, boll weevil; Aphisgossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper;Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris,tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper;Melanoplus differentialis, differential grasshopper; Thrips tabaci,onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychuscinnabarinus, carmine spider mite; Tetranychus urticae, twospottedspider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodopterafrugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspisbrunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil;Sitophilus oryzae, rice weevil; Nephotettix nigropictus, riceleafhopper; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Soybean: Pseudoplusia includens, soybeanlooper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypenascabra, green cloverworm; Ostrinia nubilalis, European corn borer;Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm;Heliothis virescens, cotton budworm; Helicoverpa zea, cotton bollworm;Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peachaphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, greenstink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Hylemya platura, seedcornmaggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onionthrips; Tetranychus turkestani, strawberry spider mite; Tetranychusurticae, twospotted spider mite; Barley: Ostrinia nubilalis, Europeancorn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum,greenbug; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Euschistus servus, brown stink bug; Deliaplatura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobialatens, brown wheat mite; Oil Seed Rape: Brevicoryne brassicae, cabbageaphid; Phyllotreta cruciferae, Flea beetle; Mamestra configurata, Berthaarmyworm; Plutella xylostella, Diamond-back moth; Delia spp., Rootmaggots.

The methods of the invention can be used with other methods available inthe art for enhancing disease resistance in plants. Similarly, theantimicrobial compositions described herein may be used alone or incombination with other nucleotide sequences, polypeptides, or agents toprotect against plant diseases and pathogens. Although any one of avariety of second nucleotide sequences may be utilized, specificembodiments of the invention encompass those second nucleotide sequencesthat, when expressed in a plant, help to increase the resistance of aplant to pathogens.

Proteins, peptides, and lysozymes that naturally occur in insects(Jaynes et al. (1987) Bioassays 6:263-270), plants (Broekaert et al.(1997) Critical Reviews in Plant Sciences 16:297-323), animals (Vunnamet al. (1997) J. Peptide Res. 49:59-66), and humans (Mitra and Zang(1994) Plant Physiol. 106:977-981; Nakajima et al. (1997) Plant CellReports 16:674-679) are also a potential source of plant diseaseresistance. Examples of such plant resistance-conferring sequencesinclude those encoding sunflower rhoGTPase-Activating Protein (rhoGAP),lipoxygenase (LOX), Alcohol Dehydrogenase (ADH), andSclerotinia-Inducible Protein-I (SCIP-1) described in U.S. Pat. No.6,709,865, issued Mar. 23, 2004, herein incorporated by reference. Thesenucleotide sequences enhance plant disease resistance through themodulation of development, developmental pathways, and the plantpathogen defense system. Other plant defense proteins include thosedescribed in WO 99/43823 and WO 99/43821, all of which are hereinincorporated by reference. It is recognized that such second nucleotidesequences may be used in either the sense or antisense orientationdepending on the desired outcome.

In another embodiment, the defensins comprise isolated polypeptides ofthe invention. The defensins of the invention find use in thedecontamination of plant pathogens during the processing of grain foranimal or human food consumption; during the processing of feedstuffs,and during the processing of plant material for silage. In thisembodiment, the defensins of the invention are presented to grain, plantmaterial for silage, or a contaminated food crop, or during anappropriate stage of the processing procedure, in amounts effective forantimicrobial activity. The compositions can be applied to theenvironment of a plant pathogen by, for example, spraying, atomizing,dusting, scattering, coating or pouring, introducing into or on thesoil, introducing into irrigation water, by seed treatment, or dustingat a time when the plant pathogen has begun to appear or before theappearance of pests as a protective measure. It is recognized that anymeans that bring the defensive agent polypeptides in contact with theplant pathogen can be used in the practice of the invention.

Additionally, the compositions can be used in formulations used fortheir antimicrobial activities. Methods are provided for controllingplant pathogens comprising applying a decontaminating amount of apolypeptide or composition of the invention to the environment of theplant pathogen. The polypeptides of the invention can be formulated withan acceptable carrier into a composition(s) that is, for example, asuspension, a solution, an emulsion, a dusting powder, a dispersiblegranule, a wettable powder, an emulsifiable concentrate, an aerosol, animpregnated granule, an adjuvant, a coatable paste, and alsoencapsulations in, for example, polymer substances.

Such compositions disclosed above may be obtained by the addition of asurface-active agent, an inert carrier, a preservative, a humectant, afeeding stimulant, an attractant, an encapsulating agent, a binder, anemulsifier, a dye, a UV protectant, a buffer, a flow agent orfertilizers, micronutrient donors or other preparations that influenceplant growth. One or more agrochemicals including, but not limited to,herbicides, insecticides, fungicides, bacteriocides, nematocides,molluscicides, acaracides, plant growth regulators, harvest aids, andfertilizers, can be combined with carriers, surfactants, or adjuvantscustomarily employed in the art of formulation or other components tofacilitate product handling and application for particular targetmycotoxins. Suitable carriers and adjuvants can be solid or liquid andcorrespond to the substances ordinarily employed in formulationtechnology, e.g., natural or regenerated mineral substances, solvents,dispersants, wetting agents, tackifiers, binders, or fertilizers. Theactive ingredients of the present invention are normally applied in theform of compositions and can be applied to the crop area or plant to betreated, simultaneously or in succession, with other compounds. In someembodiments, methods of applying an active ingredient of the presentinvention or an agrochemical composition of the present invention (whichcontains at least one of the proteins of the present invention) arefoliar application, seed coating, and soil application.

Suitable surface-active agents include, but are not limited to, anioniccompounds such as a carboxylate of, for example, a metal; a carboxylateof a long chain fatty acid; an N-acylsarcosinate; mono or di-esters ofphosphoric acid with fatty alcohol ethoxylates or salts of such esters;fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecylsulfate, or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates;ethoxylated alkylphenol sulfates; lignin sulfonates; petroleumsulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates orlower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;salts of sulfonated naphthalene-formaldehyde condensates; salts ofsulfonated phenol-formaldehyde condensates; more complex sulfonates suchas the amide sulfonates, e.g., the sulfonated condensation product ofoleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g.,the sodium sulfonate or dioctyl succinate. Non-ionic agents includecondensation products of fatty acid esters, fatty alcohols, fatty acidamides or fatty-alkyl- or alkenyl-substituted phenols with ethyleneoxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fattyacid esters, condensation products of such esters with ethylene oxide,e.g. polyoxyethylene sorbitar fatty acid esters, block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Examples of a cationic surface-active agent include, for instance, analiphatic mono-, di-, or polyamine such as an acetate, naphthenate, oroleate; or oxygen-containing amine such as an amine oxide ofpolyoxyethylene alkylamine; an amide-linked amine prepared by thecondensation of a carboxylic acid with a di- or polyamine; or aquaternary ammonium salt.

Examples of inert materials include, but are not limited to, inorganicminerals such as kaolin, phyllosilicates, carbonates, sulfates,phosphates, or botanical materials such as cork, powdered corncobs,peanut hulls, rice hulls, and walnut shells.

The compositions of the present invention can be in a suitable form fordirect application or as a concentrate of a primary composition, whichrequires dilution with a suitable quantity of water or other diluentbefore application. The decontaminating concentration will varydepending upon the nature of the particular formulation, specifically,whether it is a concentrate or to be used directly.

In a further embodiment, the compositions, as well as the polypeptidesof the present invention can be treated prior to formulation to prolongthe activity when applied to the environment of a plant pathogen as longas the pretreatment is not deleterious to the activity. Such treatmentcan be by chemical and/or physical means as long as the treatment doesnot deleteriously affect the properties of the composition(s). Examplesof chemical reagents include, but are not limited to, halogenatingagents; aldehydes such as formaldehyde and glutaraldehyde;anti-infectives, such as zephiran chloride; alcohols, such asisopropanol and ethanol; and histological fixatives, such as Bouin'sfixative and Helly's fixative (see, for example, Humason (1967) AnimalTissue Techniques (W.H. Freeman and Co.)).

In an embodiment of the invention, the compositions of the inventioncomprise a microbe having stably integrated the nucleotide sequence of adefensive agent. The resulting microbes can be processed and used as amicrobial spray. Any suitable microorganism can be used for thispurpose. See, for example, Gaertner et al. (1993) in Advanced EngineeredPesticides, Kim (Ed.). In one embodiment, the nucleotide sequences ofthe invention are introduced into microorganisms that multiply on plants(epiphytes) to deliver the defensins to potential target crops.Epiphytes can be, for example, gram-positive or gram-negative bacteria.

It is further recognized that whole, i.e., unlysed, cells of thetransformed microorganism can be treated with reagents that prolong theactivity of the polypeptide produced in the microorganism when themicroorganism is applied to the environment of a target plant. Asecretion signal sequence may be used in combination with the gene ofinterest such that the resulting enzyme is secreted outside themicroorganism for presentation to the target plant.

In this manner, a gene encoding a defensive agent of the invention maybe introduced via a suitable vector into a microbial host, and saidtransformed host applied to the environment, plants, or animals.Microorganism hosts that are known to occupy the “phytosphere”(phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one ormore crops of interest may be selected for transformation. Thesemicroorganisms are selected so as to be capable of successfullycompeting in the particular environment with the wild-typemicroorganisms, to provide for stable maintenance and expression of thegene expressing the detoxifying polypeptide, and for improved protectionof the proteins of the invention from environmental degradation andinactivation.

Such microorganisms include bacteria, algae, and fungi. Illustrativeprokaryotes, both Gram-negative and -positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiaceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such asPseudomonas and Acetobacter; Azotobacteraceae; and Nitrobacteraceae.Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, whichincludes yeast, such as Saccharomyces and Schizosaccharomyces; andBasidiomycetes yeast, such as Rhodotorula, Aureobasidium,Sporobolomyces, and the like.

Of particlular interest are microorganisms, such as bacteria, e.g.,Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces,Rhizobium, Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes;fungi, particularly yeast, e.g., Saccharomyces, Pichia, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, Aureobasidium, andGliocladium. Of particular interest are such phytosphere bacterialspecies as Pseudomonas syringae, Pseudomonas fluorescens, Serratiamarcescens, Acetobacter xylinum, Agrobacteria, Rhodopseudomonasspheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenesentrophus, Clavibacter xyli, and Azotobacter vinlandii; and phytosphereyeast species such as Rhodotorula rubra, R. glutinis, R. marina, R.aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii,Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomycesroseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pullulans.

In an embodiment of the invention, the defensins of the invention may beused as a pharmaceutical compound for treatment of fungal and microbialpathogens in humans and other animals. Diseases and disorders caused byfungal and microbial pathogens include but are not limited to fungalmeningoencephalitis, superficial fungal infections, ringworm, Athlete'sfoot, histoplasmosis, candidiasis, thrush, coccidioidoma, pulmonarycryptococcus, trichosporonosis, piedra, tinea nigra, fungal keratitis,onychomycosis, tinea capitis, chromomycosis, aspergillosis,endobronchial pulmonary aspergillosis, mucormycosis,chromoblastomycosis, dermatophytosis, tinea, fusariosis, pityriasis,mycetoma, pseudallescheriasis, and sporotrichosis.

In particular, the compositions of the invention may be used aspharmaceutical compounds to provide treatment for diseases and disordersassociated with, but not limited to, the following fungal pathogens:Histoplasma capsulatum, Candida spp. (C. albicans, C. tropicalis, C.parapsilosis, C. guilliermondii, C. glabrata/Torulopsis glabrata, C.krusei, C. lusitaniae), Aspergillus fumigatus, A. flavus, A. niger,Rhizopus spp., Rhizomucor spp., Cunninghamella spp., Apophysomyces spp.,Saksenaee spp., Mucor spp., and Absidia spp. Efficacy of thecompositions of the invention as anti-fungal treatments may bedetermined through anti-fungal assays known to one in the art.

The defensins may be administered to a patient through numerous means.Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art. Thecompounds can also be prepared in the form of suppositories (e.g., withconventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated with each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. Depending on thetype and severity of the disease, about 1 μg/kg to about 20 mg/kg (e.g.,0.1 to 20 mg/kg) of active compound is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to about 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays. Anexemplary dosing regimen is disclosed in WO 94/04188. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

“Treatment” is herein defined as the application or administration of atherapeutic agent to a patient, or application or administration of atherapeutic agent to an isolated tissue or cell line from a patient, whohas a disease, a symptom of disease or a predisposition toward adisease, with the purpose to cure, heal, alleviate, relieve, alter,remedy, ameliorate, improve or affect the disease, the symptoms ofdisease or the predisposition toward disease. A “therapeutic agent”comprises, but is not limited to, the small molecules, peptides,antibodies, and antisense oligonucleotides of the invention.

The defensins of the invention can be used for any application includingcoating surfaces to target microbes. In this manner, target microbesinclude human pathogens or microorganisms. Surfaces that might be coatedwith the defensins of the invention include carpets and sterile medicalfacilities. Polymer bound polypeptides of the invention may be used tocoat surfaces. Methods for incorporating compositions with antimicrobialproperties into polymers are known in the art. See U.S. Pat. No.5,847,047 herein incorporated by reference.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, various modifications of the invention, in addition tothose shown and described herein, will be apparent to those skilled inthe art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

The disclosure of each reference set forth herein is incorporated hereinby reference in its entirety.

Example 1 Composition of cDNA Libraries, Isolation and Sequencing ofcDNA Clones

cDNA libraries representing mRNAs from various Dimorphotheca sinuata,Picramnia pentandra, Parthenium argentatum Grey, Vernonia mespilifolia,Nicotiana benthamiana, and Helianthus annuus tissues were prepared. Thecharacteristics of the libraries are described below. TABLE 2 cDNALibraries from Dimorphotheca sinuata, Picramnia pentandra, Partheniumargentatum Grey, and Nicotiana benthamiana, Vernonia, and Helianthusannuus Library Tissue Clone dms2c African Daisy (Dimorphotheca sinuata)dms2c.pk001.d3 Developing Seed epb1c Parthenium argentatum Grey StemBark epb1c.pk001.h15 epb1c.pk002.h2 epb1c.pk003.p14 epb1c.pk004.p22epb1c.pk005.o6 epb1c.pk006.k15 epb3c Parthenium argentatum Grey StemBark epb3c.pk009.j22 pps Florida Bitterbush (Picramnia pentandra)pps.pk0011.a9 Developing Seed pps.pk0010.g2 tdr1c Nicotiana benthamianaDeveloping Root tdr1c.pk002.g7 vs1n Vernonia Seed* vs1n.pk0009.h6vs1n.pk007.a9 hss1c Sunflower plants (Helianthus annuus) hss1c.pk018.k14infected with the plant fungus Sclerotinia*This library was normalized essentially as described in U.S. Pat. No.5,482,845, incorporated herein by reference.

cDNA libraries may be prepared by any one of many methods available. Forexample, the cDNAs may be introduced into plasmid vectors by firstpreparing the cDNA libraries in Uni-ZAP™ XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).The Uni-ZAP™ XR libraries are converted into plasmid libraries accordingto the protocol provided by Stratagene. Upon conversion, cDNA insertswill be contained in the plasmid vector pBluescript. In addition, thecDNAs may be introduced directly into precut Bluescript II SK(+) vectors(Stratagene) using T4 DNA ligase (New England Biolabs), followed bytransfection into DH10B cells according to the manufacturer's protocol(GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors,plasmid DNAs are prepared from randomly picked bacterial coloniescontaining recombinant pBluescript plasmids, or the insert cDNAsequences are amplified via polymerase chain reaction using primersspecific for vector sequences flanking the inserted cDNA sequences.Amplified insert DNAs or plasmid DNAs are sequenced in dye-primersequencing reactions to generate partial cDNA sequences (expressedsequence tags or “ESTs”; see Adams et al. (1991) Science 252:1651-1656).The resulting ESTs are analyzed using a Perkin Elmer Model 377fluorescent sequencer.

Full-insert sequence (FIS) data is generated utilizing a modifiedtransposition protocol. Clones identified for FIS are recovered fromarchived glycerol stocks as single colonies, and plasmid DNAs areisolated via alkaline lysis. Isolated DNA templates are reacted withvector primed M13 forward and reverse oligonucleotides in a PCR-basedsequencing reaction and loaded onto automated sequencers. Confirmationof clone identification is performed by sequence alignment to theoriginal EST sequence from which the FIS request is made.

Confirmed templates are transposed via the Primer Island transpositionkit (PE Applied Biosystems, Foster City, Calif.), that is based upon theSaccharomyces cerevisiae Tyl transposable element (Devine and Boeke(1994) Nucleic Acids Res. 22:3765-3772). The in vitro transpositionsystem places unique binding sites randomly throughout a population oflarge DNA molecules. The transposed DNA is then used to transform DH10Belectro-competent cells (Gibco BRL/Life Technologies, Rockville, Md.)via electroporation. The transposable element contains an additionalselectable marker (named DHFR; Fling and Richards (1983) Nucleic AcidsRes. 11:5147-5158), allowing for dual selection on agar plates of onlythose subclones containing the integrated transposon. Multiple subclonesare randomly selected from each transposition reaction, plasmid DNAs areprepared via alkaline lysis, and templates are sequenced (ABI Prismdye-terminator ReadyReaction mix) outward from the transposition eventsite, utilizing unique primers specific to the binding sites within thetransposon.

Sequence data is collected (ABI Prism Collections) and assembled usingPhred/Phrap (P. Green, University of Washington, Seattle). Phrep/Phrapis a public domain software program which re-reads the ABI sequencedata, re-calls the bases, assigns quality values, and writes the basecalls and quality values into editable output files. The Phrap sequenceassembly program uses these quality values to increase the accuracy ofthe assembled sequence contigs. Assemblies are viewed by the Consedsequence editor (D. Gordon, University of Washington, Seattle).

Example 2 Identification of cDNA Clones

cDNA clones encoding plant defensin were identified by conducting BLAST(Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol.215:403-410) searches for similarity to sequences contained in the BLAST“nr” database (comprising all non-redundant GenBank CDS translations,sequences derived from the 3-dimensional structure Brookhaven ProteinData Bank, the last major release of the SWISS-PROT protein sequencedatabase, EMBL, and DDBJ databases). The cDNA sequences obtained inExample 1 were analyzed for similarity to all publicly available DNAsequences contained in the “nr” database using the BLASTN algorithmprovided by the National Center for Biotechnology Information (NCBI).The DNA sequences were translated in all reading frames and compared forsimilarity to all publicly available protein sequences contained in the“nr” database using the BLASTX algorithm (Gish and States (1993) Nat.Genet. 3:266-272) provided by the NCBI. For convenience, the P-value(probability) of observing a match of a cDNA sequence to a sequencecontained in the searched databases merely by chance as calculated byBLAST are reported herein as “pLog” values, which represent the negativeof the logarithm of the reported P-value. Accordingly, the greater thepLog value, the greater the likelihood that the cDNA sequence and theBLAST “hit” represent homologous proteins.

ESTs submitted for analysis are compared to the GenBank database asdescribed above. ESTs that contain sequences more 5′ or 3′ can be foundby using the BLASTn algorithm (Altschul et al. (1997) Nucleic Acids Res.25:3389-3402.) against the Du Pont proprietary database comparingnucleotide sequences that share common or overlapping regions ofsequence homology. Where common or overlapping sequences exist betweentwo or more nucleic acid fragments, the sequences can be assembled intoa single contiguous nucleotide sequence, thus extending the originalfragment in either the 5′ or 3′ direction. Once the most 5′ EST isidentified, its complete sequence can be determined by Full InsertSequencing as described in Example 1. Homologous genes belonging todifferent species can be found by comparing the amino acid sequence of aknown gene (from either a proprietary source or a public database)against an EST database using the tBLASTn algorithm. The tBLASTnalgorithm searches an amino acid query against a nucleotide databasethat is translated in all 6 reading frames. This search allows fordifferences in nucleotide codon usage between different species, and forcodon degeneracy.

Example 3 Characterization of cDNA Clones Encoding Plant Defensin

The BLASTX search using the EST sequences from clones dms2c.pk001.d3,epblc.pk002.h2, pps.pk0011.a9 revealed similarity of the proteinsencoded by the cDNAs to defensin from Dahlia merckii (NCBI GenBankIdentifier (GI) No. 2147320). The BLAST results for each of these ESTsare shown in Table 3: TABLE 3 BLAST Results for Clones EncodingPolypeptides Homologous to Plant Defensin BLAST pLog Clone Score 2147320dms2c.pk001.d3 30.4 epb1c.pk002.h2 30.3 pps.pk0011.a9 24.0

The BLASTX search using the EST sequence from clone tdr1c.pk002.g7revealed similarity of the protein encoded by the cDNA to defensin fromNicotiana tabacum (NCBI GI No. 676882) with a pLog value of 16.4.

The sequence of a portion of the cDNA insert from clone dms2c.pk001.d3is shown in SEQ ID NO:1; the deduced amino acid sequence of this cDNA isshown in SEQ ID NO:2. The sequence of a portion of the cDNA insert fromclone epb1c.pk002.h2 is shown in SEQ ID NO:9; the deduced amino acidsequence of this cDNA is shown in SEQ ID NO: 10. The sequence of aportion of the cDNA insert from clone pps.pk0011.a9 is shown in SEQ IDNO:5; the deduced amino acid sequence of this cDNA is shown in SEQ IDNO:6. The sequence of a portion of the cDNA insert from clonetdr1c.pk002.g7 is shown in SEQ ID NO:25; the deduced amino acid sequenceof this cDNA is shown in SEQ ID NO:26. BLAST scores and probabilitiesindicate that the instant nucleic acid fragments encode portions ofplant defensins. These sequences represent the first sequences encodingdefensin from African daisy (Dimorphotheca sinuata), Parthenium(Parthenium argentatum Grey), Florida bitterbush (Picramnia pentandra),and tobacco (Nicotiana benthamiana).

The BLASTX search using the EST sequences from clones listed in Table 4revealed similarity of the polypeptides encoded by the cDNAs to adefensin from Dahlia merckii (NCBI GenBank Identifier (GI) No. 2147320;WO 99/02038-A1; Osborn et al. (1995) FEBS Lett. 368:257-262). Shown inTable 4 are the BLAST results for individual ESTs (“EST”), the sequencesof the entire cDNA inserts comprising the indicated cDNA clones (“FIS”),contigs assembled from two or more ESTs (“Contig”), contigs assembledfrom an FIS and one or more ESTs (“Contig*”), or sequences encoding at aminimum the entire mature protein derived from an EST, an FIS, a contig,or an FIS and PCR (“CGS”): TABLE 4 BLAST Results for Sequences EncodingPolypeptides Homologous to Plant Defensin BLAST pLog Clone Status Score2147320 dms2c.pk001.d3(FIS) CGS 25.70 pps.pk0011.a9(FIS) CGS 23.10epb1c.pk002.h2(FIS) CGS 25.22 epb1c.pk001.h15(EST) CGS 25.00epb1c.pk003.p14(EST) CGS 30.22 epb1c.pk004.p22(EST) CGS 30.22epb1c.pk005.o6(EST) CGS 30.22 epb1c.pk006.k15(EST) CGS 30.22epb3c.pk009.j22(EST) CGS 30.22 tdr1c.pk002.g7(FIS) CGS 11.70pps.pk0010.g2(FIS) CGS 23.10 vs1n.pk0009.h6(FIS) CGS 27.05

The data in Table 5 represents a calculation of the percent identity ofthe amino acid sequences set forth in SEQ ID NSs:4, 8, 12, 14, 16, 18,20, 22, 24, 28, 30, and 32, and the Dahlia merckii sequence (NCBIGenBank Identifier (GI) No. 2147320; SEQ ID NO:33). TABLE 5 PercentIdentity of Amino Acid Sequences Deduced From the Nucleotide Sequencesof cDNA Clones Encoding Polypeptides Homologous to Plant DefensinPercent Identity to NCBI GenBank Identifier (GI) No. SEQ ID NO. 2147320;SEQ ID NO: 33 4 92.0 8 84.0 12 92.0 14 92.0 16 92.0 18 92.0 20 92.0 2292.0 24 92.0 28 56.0 30 84.0 32 96.0

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode entire plant defensins. These sequences represent the firstAfrican daisy (Dimorphotheca sinuata), Parthenium (Parthenium argentatumGrey), Florida bitterbush (Picramnia pentandra), tobacco (Nicotianabenthamiana), and Vernonia mespilifolia sequences encoding plantdefensin known to Applicants.

Sequence alignments and percent identity calculations were alsoperformed using the GAP program in the Wisconsin Genetics Softwarepackage, Version 10.0 (available from Genetics Computer Group, 575Science Drive, Madison, Wis., USA). The Dahlia merkii sequence (NCBIGenBank Identifier (GI) No. 2147320; SEQ ID NO: 33) was compared to SEQID NOS: 47 and 49 using the GAP program, and the percent identity valuesobtained are shown in Table 6. TABLE 6 Percent Identity of Amino AcidSequences Deduced From the Nucleotide Sequences of cDNA Clones EncodingPolypeptides Homologous to Plant Defensin Percent Identity to NCBIGenBank Identifier (GI) No. SEQ ID NO. 2147320; SEQ ID NO: 33 47 92.0 4998.0

Example 4 Expression of Chimeric Genes in Monocot Cells

A chimeric gene comprising a cDNA encoding the instant polypeptide insense orientation with respect to the maize 27 kD zein promoter that islocated 5′ to the cDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, is constructed. The cDNA fragment ofthis gene is generated by polymerase chain reaction (PCR) of the cDNAclone using appropriate oligonucleotide primers. Cloning sites (NcoI orSmaI) are incorporated into the oligonucleotides to provide properorientation of the DNA fragment when inserted into the digested vectorpML103 as described below. Amplification is then performed in a standardPCR. The amplified DNA is then digested with restriction enzymes NcoIand SmaI and fractionated on an agarose gel. The appropriate band isisolated from the gel and combined with a 4.9 kb NcoI-SmaI fragment ofthe plasmid pML103. Plasmid pML103 has been deposited under the terms ofthe Budapest Treaty at ATCC (American Type Culture Collection, 10801University Blvd., Manassas, Va. 20110-2209), and bears accession numberATCC 97366. The DNA segment from pML103 contains a 1.05 kb SalI-NcoIpromoter fragment of the maize 27 kD zein gene and a 0.96 kb SmaI-SalIfragment from the 3′ end of the maize 10 kD zein gene in the vectorpGem9Zf(+) (Promega). Vector and insert DNA is ligated at 15° C.overnight, essentially as described (Maniatis). The ligated DNA is thenused to transform E. coli XL1-Blue (Epicurian Coli XL-1 Blue™;Stratagene). Bacterial transformants are screened by restriction enzymedigestion of plasmid DNA and limited nucleotide sequence analysis usingthe dideoxy chain termination method (Sequenase™ DNA Sequencing Kit;U.S. Biochemical). The resulting plasmid construct comprises a chimericgene encoding, in the 5′ to 3′ direction, the maize 27 kD zein promoter,a cDNA fragment encoding the instant polypeptide, and the 10 kD zein 3′region.

The chimeric gene described above is then introduced into corn cells bythe following procedure. Immature corn embryos are dissected fromdeveloping caryopses derived from crosses of the inbred corn lines H99and LH132. The embryos are isolated 10 to 11 days after pollination whenthey are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept inthe dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant is cultured on N6 medium and sub-cultured on this medium every 2to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) is used in transformation experiments in order toprovide for a selectable marker. This plasmid contains the Pat gene (seeEuropean Patent Publication 0 242 236) that encodes phosphinothricinacetyl transferase (PAT). The enzyme PAT confers resistance toherbicidal glutamine synthetase inhibitors such as phosphinothricin. ThePat gene in p35S/Ac is under the control of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812) andthe 3′ region of the nopaline synthase gene from the T-DNA of the Tiplasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73)is used to transfer genes to the callus culture cells. According to thismethod, gold particles (1 μm in diameter) are coated with DNA using thefollowing technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles is then placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/Heinstrument (Bio-Rad Instruments, Hercules Calif.), using a heliumpressure of 1000 psi, a gap distance of 0.5 cm and a flying distance of1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcoveres a circular area of about 5 cm in diameter. The petri dishcontaining the tissue is placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue is transferred to N6 medium thatcontains glufosinate (2 mg per liter) and lacks casein or proline. Thetissue continues to grow slowly on this medium. After an additional 2weeks the tissue is transferred to fresh N6 medium containingglufosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus is identified on some of the plates containing theglufosinate-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 5 Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) is used for expression ofthe instant polypeptides in transformed soybean. The phaseolin cassetteincludes about 500 nucleotides upstream (5′) from the translationinitiation codon and about 1650 nucleotides downstream (3′) from thetranslation stop codon of phaseolin. Between the 5′ and 3′ regions arethe unique restriction endonuclease sites Nco I (which includes the ATGtranslation initiation codon), Sma I, Kpn I and Xba I. The entirecassette is flanked by Hind III sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites are incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

Soybean embryos are then transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, are culturedin the light or dark at 26° C. on an appropriate agar medium for 6-10weeks. Somatic embryos that produce secondary embryos are then excisedand placed into a suitable liquid medium. After repeated selection forclusters of somatic embryos that multiplied as early, globular stagedembryos, the suspensions are maintained as described below.

Soybean embryogenic suspension cultures are maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures are then transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS 1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the ³⁵S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptide and the phaseolin3′ region can be isolated as a restriction fragment. This fragment isthen inserted into a unique restriction site of the vector carrying themarker gene.

To 50 μL of a 60 mg/mL 1 μm (in diameter) gold particle suspension isadded (in order): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50μL CaCl₂ (2.5 M). The particle preparation is then agitated for threeminutes, spun in a microfuge for 10 seconds and the supernatant removed.The DNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensionis sonicated three times for one second each. Five μL of the DNA-coatedgold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue is divided in half and placed back into liquid and cultured asdescribed above.

Five to seven days post bombardment, the liquid media is exchanged withfresh media, and eleven to twelve days post bombardment with fresh mediacontaining 50 mg/mL hygromycin. This selective media is refreshedweekly. Seven to eight weeks post bombardment, green, transformed tissuemay be observed growing from untransformed, necrotic embryogenicclusters. Isolated green tissue is removed and inoculated intoindividual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line is treated as anindependent transformation event. These suspensions are then subculturedand maintained as clusters of immature embryos or regenerated into wholeplants by maturation and germination of individual somatic embryos.

Example 6 Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression vector pBT430. This vector is a derivative of pET-3a(Rosenberg et al. (1987) Gene 56:125-135), that employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA is appropriately digested to release anucleic acid fragment encoding the protein. This fragment is thenpurified on a 1% NuSieve GTG™ low melting agarose gel (FMC). Buffer andagarose contain 10 μg/ml ethidium bromide for visualization of the DNAfragment. The fragment is then purified from the agarose gel bydigestion with GELase™ (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters is purified fromthe excess adapters using low melting agarose as described above. Thevector pBT430 is digested, dephosphorylated with alkaline phosphatase(NEB) and deproteinized with phenol/chloroform as described above. Theprepared vector pBT430 and fragment is then ligated at 16° C. for 15hours followed by transformation into DH5 electrocompetent cells (GIBCOBRL). Transformants are selected on agar plates containing LB media and100 μg/mL ampicillin. Transformants containing the gene encoding theinstant polypeptide are then screened for the correct orientation withrespect to the T7 promoter by restriction enzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation continued for 3 h at 25° C. Cellsare then harvested by centrifugation and re-suspended in 50 μL of 50 mMTris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads are addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture is separated by SDS-polyacrylamide gelelectrophoresis. Gels are observed for protein bands migrating at theexpected molecular weight.

Example 7 Assaying Plant Defensin Activity

The polypeptides described herein may be produced using any number ofmethods known to those skilled in the art. Such methods include, but arenot limited to, expression in bacteria as described in Example 6, orexpression in eukaryotic cell culture, in planta, and using viralexpression systems in suitably infected organisms or cell lines. Theinstant polypeptides may be expressed either as mature forms of theproteins as observed in vivo or as fusion proteins by covalentattachment to a variety of enzymes, proteins or affinity tags. Commonfusion protein partners include glutathione S-transferase (“GST”),thioredoxin (“Trx”), maltose binding protein, and C- and/or N-terminalhexahistidine polypeptide (“(His)₆”). The fusion proteins may beengineered with a protease recognition site at the fusion point so thatfusion partners can be separated by protease digestion to yield intactmature enzyme. Examples of such proteases include thrombin, enterokinaseand factor Xa. However, any protease can be used which specificallycleaves the peptide connecting the fusion protein and the enzyme.

Purification of the instant polypeptides, if desired, may utilize anynumber of separation technologies familiar to those skilled in the artof protein purification. Examples of such methods include, but are notlimited to, homogenization, filtration, centrifugation, heatdenaturation, ammonium sulfate precipitation, desalting, pHprecipitation, ion exchange chromatography, hydrophobic interactionchromatography and affinity chromatography, wherein the affinity ligandrepresents a substrate, substrate analog or inhibitor. When the instantpolypeptides are expressed as fusion proteins, the purification protocolmay include the use of an affinity resin that is specific for the fusionprotein tag attached to the expressed enzyme or an affinity resincontaining ligands which are specific for the enzyme. For example, theinstant polypeptides may be expressed as a fusion protein coupled to theC-terminus of thioredoxin. In addition, a (His)₆ peptide may beengineered into the N-terminus of the fused thioredoxin moiety to affordadditional opportunities for affinity purification. Other suitableaffinity resins could be synthesized by linking the appropriate ligandsto any suitable resin such as Sepharose-4B. In an alternate embodiment,a thioredoxin fusion protein may be eluted using dithiothreitol;however, elution may be accomplished using other reagents which interactto displace the thioredoxin from the resin. These reagents includeβ-mercaptoethanol or other reduced thiols. The eluted fusion protein maybe subjected to further purification by traditional means as statedabove, if desired. Proteolytic cleavage of the thioredoxin fusionprotein and the enzyme may be accomplished after the fusion protein ispurified or while the protein is still bound to a ThioBond™ affinityresin (Invitrogen Corporation, Carlsbad, Calif.) or other resin.

Crude, partially purified or purified enzyme, either alone or as afusion protein, may be utilized in assays to verify over- orunderexpression of functional plant defensins in transgenic plants andtransformed bacterial cells. Assays may be conducted under well knownexperimental conditions which permit optimal enzymatic activity. Forexample, assays for plant defensin are presented by Thevissen et al.(1996) J. Biol. Chem. 271(25):15018-15025.

Example 8 Cloning of the Mature Peptide of the Picramnia pentandraDefensin Clone pps.pk0010.g2 (Pps-AMP1) into an E. coli. ExpressionVector

The nucleotide and amino acid sequences corresponding to the maturepeptide of clone pps.pk0010.g2, also known as Pps-AMP1 (SEQ ID NO:30),is shown below (mature sequences are set forth in SEQ ID NOS: 34 and35).  Q  R  L  C   E  R  A   S  L  T   W  S  G  N   C  G  N · 1CAAAGACTAT GTGAAAGAGC AAGCTTAACA TGGTCAGGCA ATTGTGGCAA GTTTCTGATACACTTTCTCG TTCGAATTGT ACCAGTCCGT TAACACCGTT· T  A  H   C  D  N  Q   C  R  S   W  E  H   A  Q  H  G · 51 CACTGCTCACTGTGACAACC AGTGTAGGTC ATGGGAGCAC GCACAACACG GTGACGAGTG ACACTGTTGGTCACATCCAG TACCCTCGTG CGTGTTGTGC·  A  C  H   V  R  G   G  K  H  M   C  F  C   Y  F  N 101 GAGCATGTCACGTACGAGGT GGAAAACATA TGTGCTTCTG CTACTTCAAT CTCGTACAGT GCATGCTCCACCTTTTGTAT ACACGAAGAC GATGAAGTTA  C  * 151 TGCTGA ACGACTThe nucleotide sequence encoding the mature peptide of Pps-AMP1 is setforth in SEQ ID NO:34. The nucleotide sequence (SEQ ID NO: 34) was PCRamplified from its corresponding cDNA clone, pps.pk0010.g2 (see Table1). The 5′ PCR primer incorporated an extra ATG sequence correspondingto a methionine residue immediately upstream of the mature peptidecoding sequence for expression in bacteria. The 5′ and 3′ PCR primerswere also designed to incorporate an NdeI and BamHI site, respectively,to facilitate cloning into the expression plasmid pET12a (Novagen,Madison Wis.). The resulting PCR product was TOPO-cloned into pCR2.1(Invitrogen, Carlsbad, Calif.) and sequence verified. A NdeI-BamHIfragment containing the Pps-AMP1 nucleotide sequence corresponding tothe mature Pps-AMP1 peptide, with the added methionine residue, wassubcloned from pCR2.1 into the corresponding sites of pET12a placing thePps-AMP1 nucleotide sequence encoding for the mature peptide undercontrol of the T7 promoter. The pET12a-PpsAMP1 construct was transformedinto a compatible expression host, BL21 (DE3, pLysS) (Invitrogen) orOrigami (DE3, pLysS) (Novagen) and expression of the mature Pps-AMP1peptide was induced by addition of IPTG as described in Example 6.

Example 9 Induction and Expression of the Pps-AMP1 Mature Peptide (SEQID NO: 35)

Expression of the Pps-AMP1 mature peptide (SEQ ID NO: 35) was inducedusing the following protocol. 1 liter (L) of LB broth (with 500 q/mlCarbenicillin and 34 μg/ml Chloramphenicol) was inoculated with 5 mls ofan overnight culture (containing the same concentration of antibiotics)derived from a single isolated colony of BL21- or Origami-transformedcells (from Example 8). The culture was incubated at 37° C. withvigorous shaking (225 rpm) until an OD₆₀₀ between 0.6-0.7 was reached.IPTG (isopropryl-B-D-galactopyranoside) was added to the culture to afinal concentration of 0.5 mM and the culture further incubatedovernight at 37° C. The next day Pps-AMP1 mature peptide expression wasconfirmed by the presence of inclusion bodies in the bacteria under1000× (oil emersion) magnification using a phase contrast lightmicroscope.

The induced bacteria were pelleted by centrifugation (15,000 rpm for 10min) and the pellet resuspended in 30 ml of 20 mM Tris-HCl (pH 7.5). Thebacteria were lysed by French press at an equivalent cell pressure of20,000 psi. The suspension was centrifuged at 15,000 rpm for 15 minutesto pellet inclusion bodies. Inclusion bodies were washed 2× with 100 mlof 20 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1% Triton X-100.

Example 10 Refolding and Purification of the Pps-AMP1 Mature Peptide(SEQ ID NO: 35)

Inclusion bodies from Example 9 were resuspended in 6 M Guanidinehydrochloride, 0.1 M Tris-HCl, pH 8.0, 1 mM EDTA, and 0.1 Mdithiothreitol. After shaking for two hours at low speed on an orbitalshaker at room temperature, any remaining particulate matter was removedby centrifugation or filtration.

The solubilized protein was precipitated by addition of 6 volumes of icecold acidic acetone (39:1 acetone: 1 M HCl). The suspension was allowedto sit on ice for about 1 hour and then centrifuged at 2000 g for 6-8minutes to pellet the protein. The pellet was washed twice with acidicacetone and allowed to air dry for 10 minutes before resolubilization indeionized water.

The unfolded protein was purified by reverse phase chromatography on aVydac™ C18 column (10 micron particle, 300 Angstrom pore size, Partnumber 218TP101510, Grace Vydac, Calif.) using a two step gradientconsisting of Solvent A (95% H₂O, 5% acetonitrile, 0.1% trifluoroaceticacid) and Solvent B (5% H₂O, 95% acetonitrile, 0.1% trifluoroaceticacid). The first step of the gradient was 10% to 24% Solvent B at a flowrate of 3 ml/min for 3 min. The second step was from 24% to 40% SolventB at a flow rate of 3 ml/min for 14 minutes. The protein was monitoredby absorbance at 214 nm. Prior to loading on the column, the sample wasadjusted to 1% trifluoroacetic acid and any precipitated materialremoved by centrifugation. The unfolded, reduced Pps-AMP1 mature peptideeluted at approximately 37% solvent B. Fractions corresponding to theunfolded peak of Pps-AMP1 mature protein were pooled and the proteinconcentration adjusted to 0.1 mg/mL-0.5 mg/mL by addition of 40%acetonitrile. The solution was brought to 0.1 M ammonium acetate, pH6-9, and 1.0 mM reduced glutathione and stirred at room temperatureuntil the Pps-AMP1 mature peptide was completely folded as determined byLC/MS analysis. Generally 24 hours was found to be sufficient forcomplete folding. Folded Pps-AMP1 mature peptide was purified by reversephase chromatography on a Vydac™ C18 column (10 micron particle, 300Angstrom pore size, Part number 218TP101510). The protein was elutedwith a linear gradient (Solvent A-95% H₂O, 5% acetonitrile and 0.1%trifluoroacetic acid, Solvent B-5% H₂O, 95% acetonitrile, 0.1%trifluoroacetic acid) from 10% to 60% Solvent B in 45 minutes at 3ml/min and monitored by absorbance at 214 nm. Pure, folded Pps-AMP1mature peptide was collected and freeze-dried. The freeze-dried proteincan be resolubilized in water and a protein assay performed to determineconcentration prior to bioassay.

Example 11 Bioactivity of Pps-AMP1 Mature Peptide Against FungalPathogens

The purified refolded and lyophilized Pps-AMP1 mature peptide wasresuspended in dH₂O to a final concentration of about 4 μg/μl. 12 μg ofpurified Pps-AMP1 mature peptide was added to 200 μl of ½ strengthpotato dextrose broth (PDB) containing a spore suspension of eitherFusarium verticilloides, Colletotrichum graminaria, or Neurospora crassacontaining 2500 spores/ml. This resulted in a stock solution with astarting concentration of 10 μM. A 0.5× dilution series for Pps-AMP1mature peptide from 10 μM through to about 0.005 μM was prepared byremoving 100 μl of the 10 μM stock and adding it to 100 μl of sporesuspension (2500 spores/ml), mixing thoroughly to achieve a 5 μMPps-AMP1 mature peptide concentration, transferring 100 μl of the 5 μMsuspension to a fresh 100 μl spore suspension etc., until about 0.005 μMwas reached. Two replicates per pathogen were performed. The fungalassay plate was scored for inhibition of fungal growth after a 48 hourincubation at 28° C. Inhibition of fungal growth was defined as littleto no spore germination without detectable hyphae growth.

The bioactivity of Pps-AMP1 mature peptide against these fungalpathogens is shown below. The IC90 concentration represents thatconcentration at which 90% of fungal growth is inhibited. Fungus IC90(uM) Colletotrichum 2.5 Fusarium 0.6 Neurospora 0.05

An in vitro assay was also performed in a 96 well microtiter plate todetermine the activity of Pps-AMP1 mature peptide against Sclerotiniasclerotiorum. Test inoculum was started by inoculating ½ strength potatodextrose broth (PDB) liquid with a sterile loop of hyphae from aSclerotinia culture propagated on ⅛ strength potato dextrose agar (PDA)plate. Liquid inoculum was held at room temperature (22° C.), withoutshaking, in the dark for 4 days, to allow sufficient growth of hyphae.The resulting suspension was macerated using a polytron tissue grinder.The sample was then diluted to the point of invisibility to the nakedeye (observation under microscopy at 40× indicated the presence ofhyphal fragments). Serial dilutions of Pps-AMP1 mature peptide in fungalsuspensions ranging from 10 μM to 0.005 μM were prepared as indicatedabove. As a positive control for this assay the fungal standard MBC(methyl 2-benzimidazolecarbamate) was formulated by adding 1 μl of eachstock solution (in DMSO) to 99 μl of inoculum to achieve the followingrates: 17, 5.67, 1.89, 0.63, 0.21, 0.07, 0.023, 0.008 μM. Sclerotiniahyphae only, or ½ strength PDB only, were included as negative controls.Activity was demonstrated by inhibition of hyphal growth after 24 hoursat 22° C.

The results are as follows: 1) PpsAmp1 mature peptide - (2 replicates)10 μM - total inhibition of fungal growth. 5 μM - moderate inhibiton offungal growth. 2.5 μM - 0.005 μM no inhibition of fungal growth. 2) MBCstandard - (2 replicates) 17-0.07 μM - total inhibition of fungalgrowth. 0.023 μM - moderate inhibition of fungal growth. 3) Inoculumcontrol - (2 replicates) Extensive fungal growth. 4) Media control - (2replicates) No contamination.

Example 12 Construction of Soybean Transformation Vectors

A synthetic version of Pps-AMP1 mature peptide (SEQ ID NO:35) operablylinked to a modified barley alpha amylase (BAA) signal peptide (SEQ IDNO: 50) (Rahmatullah R et al. (1989) Plant Mol. Biol. 12(1):119-121) wasconstructed with a codon-bias representative of Glycine max (see SEQ IDNO: 36 for the synthetic nucleotide molecule comprising the BAA signalpeptide operatively linked to the Pps-AMP1 mature peptide and SEQ ID NO:37 for the protein sequence corresponding to SEQ ID NO: 36). Codon usagebased on Glycine max was chosen to maximize the expression of Pps-AMP 1mature peptide in soybean plants. The codon preference selected for thePps-AMP1 mature peptide as well as the BAA signal sequence was derivedfrom the codon usage database available from the website of the KazusaDNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0812,Japan. See also Table 7. The BAA signal sequence was added to thePps-AMP1 mature peptide coding sequence to facilitate the export ofPps-AMP1 mature peptide out of the cell and into the intercellularspace.

The synthetic gene was constructed using a series of overlappingcomplementary oligonucleotides that were annealed together, Klenowtreated to repair the gaps, and PCR amplified using primerscorresponding to 5′ and 3′ ends of the synthetic gene. XhoI andKpnI/NcoI sites were incorporated into the PCR primers to facilitategene cloning. The PCR product was TOPO cloned into pCR2.1 (Invitrogen)and sequence verified. A XhoI-KpnI or a XhoI-NcoI fragment containingBAA-Pps-AMP 1 mature peptide was subcloned into the corresponding sitesof vectors containing either the soybean isoflavone synthetase 1 (IFS 1)promoter (see U.S. Ser. No. 10/104,706) or the UBIlZM-enhancer Rsyn7-syncore (UCP1) promoter (see U.S. Pat. No. 6,072,050 and U.S. Ser. No.60/329,667). This placed BAA-mature Pps-AMP1 behind these two promoterswith a 3′ sequence corresponding to either the NOS or pin II (potatoproteinase inhibitor gene; see Ryan (1990) Ann. Rev. Phytopath.28:425-449 and Duan et al. (1996) Nature Biotechnology 14:494-498)terminator sequences. IFS1 is a strong constitutive root promoter thatis also stress-inducible in aerial portions of the plant. The UCP1promoter is a constitutive promoter that is SCN-inducible. Expression ofPps-AMP1 mature peptide either constitutively or inducibly in roottissue was desirable to test its effect on SCN. Furthermore, theinducibility of the IFS1 promoter due to stress also made this promotersuitable to direct expression of Pps-AMP1 mature peptide in tissuessusceptible to Sclerotinia infection. TABLE 7 Glycine max [gbpln]: 619CDS's (241657 codons) fields: [triplet] [frequency: per thousand]([number]) UUU 20.5 (4964) UCU 17.5 (4238) UAU 15.8 (3808) UGU 7.2(1748) UUC 21.0 (5067) UCC 12.2 (2949) UAC 15.2 (3667) UGC 7.5 (1821)UUA 8.4 (2030) UCA 14.9 (3590) UAA 1.1  (256) UGA 0.9  (221) UUG 22.1(5343) UCG 4.6 (1107) UAG 0.6  (143) UGG 11.9 (2866) CUU 23.5 (5676) CCU19.8 (4794) CAU 13.5 (3254) CGU 7.0 (1697) CUC 16.8 (4053) CCC 10.1(2445) CAC 10.9 (2630) CGC 6.4 (1538) CUA 8.1 (1962) CCA 20.2 (4875) CAA20.5 (4964) CGA 4.0  (964) CUG 12.0 (2900) CCG 4.2 (1022) CAG 17.2(4147) CGG 2.8  (683) AUU 26.0 (6275) ACU 17.5 (4231) AAU 21.2 (5132)AGU 12.1 (2935) AUC 16.5 (3981) ACC 14.7 (3562) AAC 22.9 (5524) AGC 10.9(2640) AUA 12.8 (3086) ACA 14.9 (3601) AAA 26.4 (6370) AGA 14.3 (3459)AUG 22.4 (5404) ACG 4.2 (1006) AAG 37.5 (9052) AGG 13.3 (3218) GUU 26.7(6455) GCU 28.1 (6796) GAU 32.9 (7955) GGU 21.7 (5248) GUC 12.3 (2971)GCC 16.7 (4042) GAC 20.4 (4931) GGC 13.8 (3339) GUA 7.3 (1768) GCA 22.4(5421) GAA 33.9 (8194) GGA 22.5 (5434) GUG 22.1 (5342) GCG 6.1 (1470)GAG 34.3 (8296) GGG 12.8 (3097)Coding GC 46.16% 1st letter GC 53.12% 2nd letter GC 39.75% 3rd letter GC45.62%

Example 14 Soybean Embryo Transformation

Soybean embryos were bombarded with a plasmid containing the Pp s-AMP1mature peptide encoding nucleotide sequence operably linked to eitherthe IFS1 or UCP1 promoters as follows; somatic embryos derived fromcotyledons less than 4 mm in length, dissected from surface-sterilized,immature seeds of the soybean cultivar Jack, were cultured in the lightor dark at 26° C. on an appropriate agar medium for six to ten weeks.Somatic embryos producing secondary embryos were excised and placed intoa suitable liquid medium. After repeated selection for clusters ofsomatic embryos that multiplied as early, globular-staged embryos, thesuspensions were maintained as described below.

Soybean embryogenic suspension cultures were maintained in 35 ml liquidmedia on a rotary shaker at 150 rpm and 26° C. with fluorescent lightson a 16:8 hour day/night schedule. Cultures were subcultured every twoweeks by inoculating approximately 35 mg of tissue into 35 ml of liquidmedium.

Soybean embryogenic suspension cultures were then used fortransformation experiments by the method of particle gun bombardment(Klein et al. (1987) Nature (London) 327:70-73, U.S. Pat. No.4,945,050). A DuPont® Biolistic PDS1000®/HE instrument (helium retrofit)was used for these transformations.

A selectable marker expression cassette that can be used to facilitatesoybean transformation comprises the 35S promoter from CauliflowerMosaic Virus (Odell et al. (1985) Nature 313:810-812), the hygromycinphosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al.(1983) Gene 25:179-188), and the 3′ region of the nopaline synthase(NOS) gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The expression cassette comprising the nucleotide sequenceencoding the Pps-AMP1 mature peptide operably linked to a promoter canbe isolated as a restriction fragment. This fragment is then insertedinto a unique restriction site of the vector carrying the marker gene.

To 50 μl of a 60 mg/ml 1 μm (in diameter) gold particle suspension wasadded (in order): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M), and 50μl CaCl₂ (2.5 M). The particle preparation was then agitated for threeminutes, spun in a microfuge for 10 seconds and the supernatant removed.The DNA-coated particles were then washed once in 400 μl 70% ethanol andresuspended in 40 μl of anhydrous ethanol. The DNA/particle suspensionwas sonicated three times for one second each. Five microliters of theDNA-coated gold particles were then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture was placedin an empty 60×15 mm Petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressurewas set at 1100 psi, and the chamber was evacuated to a vacuum of 28inches mercury. The tissue was placed approximately 3.5 inches away fromthe retaining screen and bombarded three times. Following bombardment,the tissue was divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media was exchanged withfresh media, and eleven to twelve days post-bombardment with fresh mediacontaining 50 mg/ml hygromycin. This selective media was refreshedweekly. Seven to eight weeks post-bombardment, green, transformed tissuewas observed growing from untransformed, necrotic embryogenic clusters.Isolated green tissue was removed and inoculated into individual flasksto generate new, clonally propagated, transformed embryogenic suspensioncultures. Each new line was treated as an independent transformationevent. These suspensions were then subcultured and maintained asclusters of immature embryos or regenerated into whole plants bymaturation and germination of individual somatic embryos.

Example 15 DNA Preparation and PCR of Pps-AMP1 Events

The presence of the nucleotide sequence encoding the Pps-AMP1 maturepeptide was confirmed in transgenic soybean events by PCR amplification.Genomic DNA was prepared from callus by shaking approximately 100 μl ofcallus at 1500 strokes/minute for 45 seconds in the Geno/Grinder in thepresence of 1 steel ball ({fraction (5/32)}″), 300 μl of urea extractionbuffer and 300 μl of phenol/chloroform/isoamyl alcohol (25:24:1). Tubeswere centrifuged at full speed for 5 minutes and 200 μl of the aqueousphase transferred to a 96 deep well block. DNA was precipitated with anequal volume of isopropanol, centrifuged at full speed for 10 minutes,and the DNA pellets washed with 70% ethanol. After a further 5 minutecentrifugation the supernatant was removed completely and the pelletsdried in a speed vacuum. The DNA was resuspended in 100 μl of 10 mM TrisHCL pH 8 and 2 μl used for PCR amplification.

PCR amplification was performed in a 96 well format using two sets ofprimers corresponding to the promoter/5′ junction and the 3′/terminatorjunction. The primer names and sequences are as follows: SEQ ID NO: 38BAA1; 5′-GCTCGAGATGGCCAACAAGCATC-3′; SEQ ID NO: 39 BAA2;5′-CACATGTGTTTGCCTCCTCTAACG-3′; SEQ ID NO: 40 UCP1-1;5′-TCCACTCGAGCGGCTATAAATACG-3′; SEQ ID NO: 41IFS1-P1;5′-CTTTGCGTCCTTGAAAAGTCCATG-3′; SEQ ID NO: 42 PIN2; 5′-GGCCAATCCAGAAGATGGACAAGT-3′; SEQ ID NO: 43 NOS1;5′-CGCAAGACCGGCAACAGGATTC-3′;

For events containing IFS1:BAA-mature Pps-AMP1; primer pairs BAA2(SEQ IDNO: 39)/IFS1-P1(SEQ ID NO:41) and BAA1(SEQ ID NO:38)/NOS1(SEQ ID NO:43)were used. Events containing UCP1:BAA-mature Pps-AMP1 were PCR confirmedwith the primer pairs BAA2(SEQ ID NO:39)/UCP1-1(SEQ ID NO:40) andBAA1(SEQ ID NO:38)/PIN2(SEQ ID NO:42).

PCR reactions contained 2 μl of genomic DNA preparation, 10 μl ofReadyTaq mix (Sigma), 0.5 μl of each primer (10 μM) of the primer pairin a total of reaction volume of 20 μl. PCR reactions were performedusing one cycle of 95° C. for 5 min, 40 cycles of 95° C. 30 sec, 60° C.1 min, 72° C. 1 min, and 1 cycle of 72° C. for 5 min. PCR products ofthe correct size were detected on a 1% agarose gel. Greater than 95% ofthe events showed PCR products corresponding to the predicted size forboth primer pairs indicating that the events tested contained theexpected construct.

Example 16 RT-PCR of IFS1:BAA-Mature Pps-AMP1 Events

Total RNA was extracted from a subset of IFS1:BAA-mature Pps-AMP1 eventsby collecting one leaflet per event into a 2 ml sterile screw cap tube,adding 2 steel balls ({fraction (5/32)}″), and 1.0 ml of Trizol Reagent(GIBCO-BRL). Leaflets were homogenized in a DNA FastPrep instrument at aspeed of 4.5 for 45 seconds and the tubes centrifuged for 10 min at 4°C. The supernatant was extracted with chloroform and the RNAprecipitated from the aqueous phase with cold isopropyl alcohol. After a10 minute centrifugation step the pellet was washed with 70% ethanol anddried in a speed vacuum. The pellet was resuspended in 90 μl ofDEPC-treated H₂O and amplification grade DNAseI (1 U) and 10×DNAse Ibuffer added to a total volume of 100 μl. The reaction was incubated at37° C. for 15 min, twice extracted with an equal volume ofphenol/chloroform/isoamyl alcohol and the RNA precipitated from theaqueous phase with 0.1 volume 3 M sodium acetate and 2.5 volume of 100%ethanol. After centrifugation the pellet was resuspended in 30 μl ofDEPC—H₂O. RNA samples were stored at −80° C. until use.

RT-PCR amplification of the Pps-AMP1 mRNA was performed using theOne-Step RT-PCR kit (GIBCO-BRL) and the gene specific primers namedIFS1:BAA-mature Pps PCR1(5′-CCCGGGCTCGAGATGGCCAACAAGCATCTTTCTCTCAGTC-3′, see also SEQ ID NO: 44)and IFS1:BAA-mature Pps PCR2(5′-CCATGGTACCTTAACAGTTAAAATAACAGAAGCACATGTG-3′, see also SEQ ID NO:45).

The reaction mixture contained 12.5 ul of 2×One-step RT-PCR reactionmix, 0.5 μl of each primer (10 μM), 0.5 μl of RT/Platinum Taq mix in atotal volume of 15 μl. RT-PCR was performed using the followingconditions: 50° C. 30 minutes, 94° C. 2 minutes followed by 35 cycles of94° C. 30 seconds, 60° C. 1 minute, 72° C. 1 minute, and one cycle of72° C. 5 minutes. RT-PCR products were visualized on a 1.5% agarose gel.

The results of these analyses demonstrated that the subset of transgenicevents tested were expressing detectable levels of Pps-Amp1 maturepeptide.

Example 17 TO SCN Bioassay of Transgenic Events Containing the MaturePps-AMP1 Peptide

Race 1 Heterodera glycines Soybean Cyst Nematodes (SCN) were used toinfest transgenic T0 soybean plants in soil. SCN egg inoculum wasacquired by harvesting cysts from plants infested 4-6 weeks earlier.Briefly, the soil was rinsed from the roots and passed through nested 20mesh and 60 mesh screens. The material retained by the 20 mesh screenwas discarded but the material retained by the 60 mesh screen was washedthoroughly and the creamy white cysts were recovered (older brown cystsare ignored). Similarly, the plant's root system was scrubbed againstthe 20 mesh screen nested over the 60 mesh screen. Cysts were harvestedfrom the debris on the 60 mesh screen. Eggs were released from the cystsby means of a dounce homogenizer in the presence of 0.5% Clorox for 2.5minutes. Following this treatment the eggs were washed with sterilewater from the homogenizer onto the surface of a 200 mesh screen. Theeggs were then rinsed in water for an additional 5 minutes. Eggs weretransferred to a 50 ml conical tube and counted. The eggs were dilutedto 5000 eggs/ml. Plants grown in 15 cm conical tubes were inoculatedwith about 5000 eggs. Plants were maintained in a 26° C. growth chamberwith 12:12 light:dark cycle for 1 month prior to harvest and counting ofcysts.

Whole plant bioassays of events containing the nucleotide sequenceencoding the mature Pps-AMP1 peptide under control of the UCP1 (see FIG.3) or IFS1 (see FIG. 4) promoters displayed a range of resistance (asdetermined by the number of cysts) relative to the negative control(transformed Jack soybean cultivar that did not contain heterologousDNA) and the wild-type Essex cultivar soybean plants. Results from asubset of plants tested are shown in FIGS. 3 and 4. The results of thefull set of plants tested appears in Tables 8 and 9. This range ofresistance (based on cyst number) is expected for the population ofevents generated since differences in Pps-AMP1 mature peptide expressionlevels may be variable depending on such parameters as, integration siteand copy number, among others. While there was a reduction in cystnumbers with both constructs compared to the negative control Jackcultivar or the Essex cultivar, in general, those events containing thePps-AMP1 mature peptide encoding nucleotide sequence operatively linkedto the IFS1 promoter (IFS1:BAA-mature Pps-AMP1) performed better thanthose events containing the Pps-AMP1 mature peptide encoding nucleotidesequence operatively linked to the UCP1 promoter (UCP1:BAA-maturePps-AMP1). Several events exhibited excellent resistance with no cystsobserved in the root systems. TABLE 8 SCN (Race1) whole plant bioassayof transgenic events containing the Pps-AMP1 mature peptide operativelylinked to the Barley Alpha Amylase signal peptide and driven by the UCP1promoter TRANSPLANT DATE: 1/31/02 INOCULATION DATE: 2/1/02 INOCULATIONMETHOD: DISPENSE 5000 SCN EGGS 1″ BELOW SOIL SURFACE NEAR ROOTSEVALUATION DATE: 3/7/02 EVENT # CYST # EVENT # CYST # 3177-2-3-1 2403178-5-2-1 76 3177-2-3-2 60 3178-5-2-2 44 3177-2-3-3 15 3178-5-2-3 273177-2-1-1 130 3178-4-2-1 110 3177-2-1-2 82 3178-4-2-2 19 3177-2-1-3 1203178-4-2-3 53 3178-4-2-4 8 3177-4-2-1 51 3178-1-1-1 107 3177-4-2-2 NOPLANT 3178-1-1-2 34 3177-4-2-3 65 3178-1-1-3 81 3177-3-3-1 1203178-5-1-1 110 3177-3-3-2 8 3178-5-1-2 220 3177-3-3-3 160 3178-5-1-3 213177-3-5-1 16 3178-6-1-1 0 3177-3-5-2 85 3178-6-1-2 102 3177-3-5-3 633178-6-1-3 0 3177-6-2-1 54 3178-4-1-1 85 3177-6-2-2 43 3178-4-1-2 33177-6-2-3 200 3178-4-1-3 25 3177-5-2-1 0 3178-1-2-1 190 3177-5-2-2 NOPLANT 3178-1-2-2 110 3177-5-2-3 125 3178-1-2-3 37 3177-1-3-1 503178-4-3-1 120 3177-1-3-2 66 3178-4-3-2 0 3177-1-3-3 144 3178-4-3-3 593177-5-4-1 26 3178-5-5-1 53 3177-5-4-2 22 3178-5-5-2 29 3177-5-4-3 1203177-2-4-1 95 3178-1-3-1 34 3177-2-4-2 55 3178-1-3-2 16 3177-2-4-3 573178-1-3-3 0 3177-5-1-1 115 ESSEX 230 3177-5-1-2 14 ESSEX 155 3177-5-1-367 ESSEX 90 3177-1-2-1 neg 225 control 3177-1-2-2 170 3177-1-2-3 62

TABLE 9 SCN (Race1) whole plant bioassay of transgenic events containingthe Pps-AMP1 mature peptide operatively linked to the Barley AlphaAmylase signal peptide and driven by the IFS promoter INOCULATION DATE:3/8/02 EVALUATION DATE: 4/8/02 INOCULATION METHOD: DISPENSE 5000 EGGS 1″BELOW SOIL SURFACE. EVENT # CYST # EVENT # CYST # 3193-6-4-1 1953193-1-1-1 128 3193-6-4-2 255 3193-1-1-2 79 3193-6-4-3 3193-1-1-3 1703192-3-1-1 10 3193-1-4-1 49 3192-3-1-2 228 3193-1-4-2 0/no roots3192-3-1-3 3193-1-4-3 83 3192-3-2-1 16 3193-1-10-1 160 3192-3-2-2 1033193-1-10-2 28 3192-3-2-3 7 3193-1-10-3 275 3192-3-4-1 23 3193-2-1-1 523192-3-4-2 0 3193-2-1-2 41 3192-3-4-3 0 3193-2-1-3 203 3192-4-1-1 2643193-2-3-1 116 3192-4-1-2 93 3193-2-3-2 203 3192-4-1-3 18 3193-2-3-3 1123192-5-3-1 3193-2-5-1 70 3192-5-3-2 0/poor roots 3193-2-5-2 103192-5-3-3 0/poor roots 3193-2-5-3 3 3192-6-1-1 37 3193-3-4-1 983192-6-1-2 311 3193-3-4-2 210 3192-6-1-3 143 3193-3-4-3 0/poor roots3192-6-8-1 189 3193-3-5-1 3/poor roots 3192-6-8-2 74 3193-3-5-2 813192-6-8-3 3193-3-5-3 64 3192-6-10-1 236 3192-6-10-2 385 3192-6-10-3 275ESSEX 110 ESSEX 207 ESSEX 99

Example 18 T0 Sclerotinia sclerotiorum Detached Leaf Bioassay ofTransgenic Events Containing the Pps-AMP1 Mature Peptide

Sclerotinia cultures were maintained on ⅛ strength potato dextrose agar(PDA) plates at room temperature in the dark. Cultures were grown byremoving 5 mm agar plugs from the maintainence plates and placing theplugs hyphae side down on new PDA plates. Cultures were allowed to growas indicated above for 3 days. Two individual leaves from 3 plants perevent were inoculated with a 5 mm agar plug removed from the growingedge of the culture. Leaves were placed bottom side up on moistenedcotton pads in 100×100 mm square Petri plates. Leaves were inoculated byplacing the plug, hyphae side down, in the center of leaf. Plates werecovered with a lid and incubated in the dark at room temperature.Evaluation of the bioassay was performed 3 days post-inoculation bymeasuring lesion size (the product of multiplying the diameter of eachlesion in 2 directions).

The results of the Sclerotinia detached leaf assay with selectedUCP1:BAA-mature Pps-AMP1 events (see FIG. 5) and selectedIFS1:BAA-mature Pps-AMP1 events (see FIG. 6) also show the expectedvariability among the events tested. Results from the full set of eventsis shown in Tables 10 and 11. Overall some reduction in lesion sizerelative to the negative control Jack cultivar or the Essex cultivarwere observed in the UCP1:BAA-mature Pps-AMP1 events. However, theeffect of PpsAMP1 mature peptide on Sclerotina infection was even moresignificant in the IFS1:BAA-mature Pps-AMP1 events. The IFS1:BAA-maturePps-AMP1 events exhibited lesion sizes that were either greatly reducedor absent altogether. Inspection of the agar plugs in those eventsshowing reduced or no lesions demonstrated that hyphal growth wasevident and in several instances were inhibited at the junction betweenagar plug and leaf. These results are consistent with the expectedrelative strength of the two promoters used since the IFS1 promoter is astrong stress-inducible promoter in aerial tissue which is presumablyresponding to infection by Sclerotinia and the UCP1 promoter is aconstitutive promoter which does not express as strongly as the IFS1promoter. TABLE 10 Detached leaf assay for transgenic events containingthe Pps-AMP1 mature peptide operatively linked to the Barley AlphaAmylase signal peptide and driven by the UCP1 promoterUCP:BAA:PPS-AMP1 - SCLEROTINIA DETACHED LEAF BIOASSAY TRANSPLANT DATE:1/31/02 INOCULATION DATE: 2/5/02 EVALUATION DATE: 2/8/02 LESION SIZE -MEASURED IN MM. LESION AREA LESION AREA LESION AREA LESION AREA EVENT #REP 1 REP 2 EVENT # REP 1 REP 2 3177-2-3-1 15 × 21 = 315 19 × 17 = 3233178-5-2-1 15 × 13 = 195 21 × 28 = 588 3177-2-3-2 18 × 18 = 324 15 × 18= 270 3178-5-2-2 32 × 21 = 672 39 × 22 = 858 3177-2-3-3 10 × 10 = 100 14× 25 = 350 3178-5-2-3 21 × 28 = 588 30 × 20 = 600 3177-2-1-1 16 × 21 =336 21 × 19 = 399 3178-4-2-1 14 × 14 = 196 18 × 17 = 306 3177-2-1-2 25 ×17 = 425 28 × 20 = 560 3178-4-2-2 27 × 16 = 432 21 × 15 = 315 3177-2-1-321 × 17 = 357 23 × 27 = 621 3178-4-2-3 18 × 33 = 594 26 × 20 = 5203178-4-2-4 20 × 17 = 340 21 × 18 = 378 3177-4-2-1 18 × 28 = 504 24 × 22= 528 3178-1-1-1 20 × 27 = 540 32 × 21 = 672 3177-4-2-2 NO PLANT3178-1-1-2 21 × 20 = 420 16 × 19 = 304 3177-4-2-3 13 × 20 = 260 18 × 30= 540 3178-1-1-3 23 × 28 = 644 27 × 27 = 729 3177-3-3-1 19 × 22 = 418 21× 21 = 441 3178-5-1-1 18 × 27 = 486 27 × 17 = 459 3177-3-3-2 20 × 16 =320 19 × 17 = 323 3178-5-1-2 20 × 28 = 560 18 × 17 = 306 3177-3-3-3 29 ×21 = 609 32 × 21 = 672 3178-5-1-3 15 × 23 = 345 17 × 17 = 289 3177-3-5-121 × 18 = 378 26 × 19 = 494 3178-6-1-1 13 × 13 = 169 12 × 14 = 1683177-3-5-2 11 × 15 = 165 18 × 18 = 324 3178-6-1-2 14 × 15 = 210 12 × 19= 228 3177-3-5-3 20 × 36 = 720 32 × 18 = 576 3178-6-1-3 20 × 18 = 360 20× 15 = 300 3177-6-2-1 18 × 29 = 522 22 × 30 = 660 3178-4-1-1 18 × 18 =324 18 × 26 = 468 3177-6-2-2 10 × 19 = 190 11 × 29 = 319 3178-4-1-2 21 ×22 = 462 19 × 14 = 266 3177-6-2-3 16 × 20 = 320 17 × 24 = 408 3178-4-1-310 × 10 = 100 22 × 11 = 242 3177-5-2-1 16 × 18 = 288 14 × 12 = 1683178-1-2-1 21 × 25 = 525 25 × 19 = 475 3177-5-2-2 21 × 27 = 567 22 × 16= 352 3178-1-2-2 13 × 13 = 169 20 × 12 = 240 3177-5-2-3 13 × 20 = 260 16× 11 = 176 3178-1-2-3 18 × 19 = 342 20 × 27 = 540 3177-1-3-1 14 × 12 =168 10 × 9 = 90 3178-4-3-1 32 × 19 = 608 15 × 23 = 345 3177-1-3-2 23 ×33 = 759 25 × 17 = 425 3178-4-3-2 14 × 14 = 196 11 × 14 = 154 3177-1-3-334 × 20 = 680 20 × 14 = 280 3178-4-3-3 19 × 20 = 380 27 × 18 = 4863177-5-4-1 17 × 22 = 374 18 × 17 = 306 3178-5-5-1 38 × 26 = 988 31 × 21= 651 3177-5-4-2 16 × 25 = 400 16 × 18 = 288 3178-5-5-2 21 × 18 = 378 17× 21 = 357 3177-5-4-3 29 × 213 = 667 28 × 20 = 560 3177-2-4-1 17 × 24 =408 15 × 21 = 315 3178-1-3-1 16 × 21 = 336 20 × 19 = 380 3177-2-4-2 13 ×19 = 247 13 × 26 = 338 3178-1-3-2 23 × 26 = 598 14 × 25 = 350 3177-2-4-312 × 17 = 204 28 × 9 = 252 3178-1-3-3 32 × 19 = 608 33 × 21 = 7143177-5-1-1 22 × 16 = 352 18 × 27 = 486 ESSEX 37 × 24 = 888 34 × 21 = 7143177-5-1-2 10 × 12 = 120 15 × 18 = 270 20 × 31 = 620 23 × 21 4833177-5-1-3 10 × 14 = 140 10 × 12 = 120 NEG CONTROLS 3177-1-2-1 19 × 26 =494 17 × 21 = 357 3177-1-2-2 25 × 22 = 550 22 × 21 = 462 3177-1-2-3 20 ×18 = 360 31 × 20 = 620

TABLE 11 Detached leaf assay for transgenic events containing thePps-AMP1 mature peptide operatively linked to the Barley Alpha Amylasesignal peptide and driven by the IFS promoter IFS:BAA:PPS-AMP1 -SCLEROTINIA DETACHED LEAF BIOASSAY INOCULATION DATE: 3/26/02 EVALUATIONDATE: 3/29/02 LESION SIZE - MEASURED IN MM. LESION AREA LESION AREALESION AREA LESION AREA EVENT # REP 1 REP 2 EVENT # REP 1 REP 23192-2-1-4 0 0 3193-1-1-4 20 0 W/SPOTS 3192-2-1-5 0 50 3193-1-1-5 0 03192-2-1-6 0 0 3193-1-1-6 70 80 3192-3-1-5 4 6 3193-1-4-4 132 903192-3-1-6 110 54 3193-1-4-5 168 168 3193-1-4-6 0 192 3192-3-2-4 0 RIB3192-3-2-5 0 0 3193-1-10-4 15 99 3192-3-2-6 SPOTS SPOTS 3193-1-10-5SPOTS 0 3193-1-10-6 0 0 3192-3-4-4 0 SPOTS 3192-3-4-5 4 120 3193-2-1-4 460 3193-2-1-5 0 0 3192-4-1-4 0 0 3193-2-1-6 0 0 3192-4-1-5 0 4503192-4-1-6 VEIN 225 3193-2-3-4 15 99 3193-2-3-5 0 0 3192-5-3-4 0 03193-2-3-6 0 0 3192-5-3-5 0 49 3193-2-5-4 260 208 3192-6-1-5 0 03193-2-5-6 0 0 3192-6-1-6 0 W/SPOTS 126 3193-3-4-4 162 156 3192-6-8-4 00 3193-3-4-5 0 0 3192-6-8-5 8 6 3193-3-4-6 0 20 3192-6-8-6 SPOTS SPOTS3193-3-5-4 150 56 3192-6-10-4 4 100 3193-3-5-5 25 25 3192-6-10-5 25 703193-3-5-6 0 0 W/SPOTS 3192-6-10-6 0 0 3193-5-1-4 0 0 ESSEX 224 1953193-5-1-5 91 117 ESSEX 100 156 ESSEX 270 110 3193-6-4-5 RIB 203193-6-4-6 0 0 NEG CONTROLS 3193-4-5-4 0 0 3193-4-5-5 0 0 3193-4-5-6 600450

Example 19 Bioassay for Testing the Pesticidal Activity of the Proteinsof the Invention Against Southern Corn Rootworm (SCRW) and Western CornRootworm (WCRW)

Bio-Serv diet (catalog number F9800B, from: BIOSERV, EntomologyDivision, One 8th Street, Suite 1, Frenchtown, N.J. 08825) is dispensedin 128-well CD International Bioassay trays (catalog number BIO-BA-128from CD International, Pitman, N.J. 08071).

Protein samples are applied topically to the diet surface. Enough samplematerial is supplied to provide for replicate observations per sample.The trays are allowed to dry. Rootworm larvae are dispensed into thewells of the bioassay trays. A lid (catalog number BIO-CV-16, CDInternational, Pitman, N.J., 08071) is placed on each tray, and thetrays are placed in an incubator at 26° C. for 4 to 7 days.

For the evaluation of pesticidal activity against SCRW and WCRW, insectsare exposed to a solution comprising either buffer (50 mM carbonatebuffer (pH 10)) or a solution of protein sample at selected doses, forexample, 50 or 5.0 μg/cm².

The bioassays are then scored by counting “live” versus “dead” larvae.Mortality is calculated as a percentage of dead larvae out of the totalnumber of larvae tested.

Example 20 Bioassay for Testing the Pesticidal Activity of the Proteinsof the Invention Against the Colorado Potato Beetle (Leptinotarsadecemlineata)

Briefly, bioassay parameters are as follows: Bio-Serv diet (catalognumber F9800B, from: BIOSERV, Entomology Division, One 8th Street, Suite1, Frenchtown, N.J. 08825) is dispensed in a 96 well microtiter plate(catalog number 353918, Becton Dickinson, Franklin Lakes, N.J.07417-1886) having a surface area of 0.33 cm². Protein samples of theinvention are applied topically to the diet surface. Enough samplematerial is supplied to provide for 8 observations/sample. After thesamples dry, 1 Colorado potato beetle neonate is added to each wellproviding for a total of 8 larvae/sample. A Mylar® lid (Clear LamPackaging, Inc., 1950 Pratt Blvd., Elk Grove Village, Ill. 60007-5993)is affixed to each tray. Bioassay trays are placed in an incubator at25° C. The test is scored for mortality on the 7th day following liveinfesting.

Example 21 Bioassay for Testing the Pesticidal Activity of the Proteinsof the Invention Against Lepidopterans

Neonate larvae are reared according to standard protocols, such as thosepublished by Czapla and Lang (1990) J. Economic Entomology 83:2480-2485. Test compounds are either applied topically to the diet orincorporated into the larvae diet (see Czapla and Lang (1990) EconomicEntomology 83; 2480-2485. The larvae diet is dispensed to bioassaytrays. One larva is applied per well of the bioassay tray. Weight andmortality are recorded 7 days following the start of the test.

Example 22 Homopteran Membrane Feeding Bioassay for Screening Proteinsof the Invention

This assay can be used for a variety of homopterans. The assay involvestrapping the sample protein between two layers of maximally stretchedparafilm which act as a sachet on top of a small vessel containing theinsect of choice.

The assay is prepared as follows: 1 cm diameter polystyrene tubing iscut into 15 mm lengths. One end of the tube is then capped with a finemesh screen. Five insects are then added to the chamber after which thefirst layer of parafilm is stretched over the remaining open end. 25 μlof sample (polypeptide in a 5% sucrose solution containing McCormickgreen food coloring) is then placed on top of the stretched parafilm. Asecond layer of parafilm is then stretched by hand and placed over thesample. The sample is spread between the two layers of parafilm to makea continuous sachet on which the insects feed. The sachet is thencovered tightly with saran wrap to prevent evaporation and produce aslightly pressurized sample. The assay tubes are monitored for insectreproduction and death on a 24 hour basis and compared to the 5% sucrosecontrol.

All publications, patents and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications, patents and patentapplications are herein incorporated by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. An isolated polypeptide having antimicrobial activity, wherein saidpolypeptide comprises an amino acid sequence having at least 80%sequence identity to SEQ ID NO: 8, 30, or
 35. 2. The polypeptide ofclaim 1, wherein said polypeptide comprises an amino acid sequencehaving at least 90% sequence identity to SEQ ID NO: 8, 30, or
 35. 3. Thepolypeptide of claim 1, wherein said polypeptide comprises an amino acidsequence having at least 95% sequence identity to SEQ ID NO: 8, 30, or35.
 4. The polypeptide of claim 1, wherein said polypeptide comprisesthe amino acid sequence set forth in SEQ ID NO: 8, 30, or
 35. 5. Thepolypeptide of claim 1, wherein said polypeptide is characterized byantimicrobial activity against at least one plant pathogen which is afungus.
 6. The polypeptide of claim 5, wherein said fungus isSclerotinia sclerotiorum.
 7. The polypeptide of claim 1, wherein saidpolypeptide is characterized by pesticidal activity against at least oneinsect pest.
 8. The polypeptide of claim 7, wherein said insect pest isa nematode.
 9. The polypeptide of claim 8, where said nematode is theSoybean Cyst Nematode.
 10. An isolated polypeptide having antimicrobialactivity, wherein said polypeptide comprises at least 15 consecutiveamino acids of the sequence set forth in SEQ ID NO: 8, 30, or
 35. 11.The polypeptide of claim 10, wherein said polypeptide comprises at least25 consecutive amino acids of the sequence set forth in SEQ ID NO: 8,30, or
 35. 12. The polypeptide of claim 10, wherein said polypeptidecomprises at least 30 consecutive amino acids of the sequence set forthin SEQ ID NO: 8, 30, or
 35. 13. The polypeptide of claim 10, whereinsaid polypeptide comprises at least 50 consecutive amino acids of thesequence set forth in SEQ ID NO: 8, 30, or
 35. 14. The polypeptide ofclaim 10, wherein said polypeptide is characterized by antimicrobialactivity against at least one plant pathogen which is a fungus.
 15. Thepolypeptide of claim 14, wherein said fungus is Sclerotiniasclerotiorum.
 16. The polypeptide of claim 10, wherein said polypeptideis characterized by pesticidal activity against at least one insectpest.
 17. The polypeptide of claim 16, wherein said insect pest is anematode.
 18. The polypeptide of claim 17, where said nematode is theSoybean Cyst Nematode.
 19. The polypeptide of claim 12, wherein saidpolypeptide is characterized by pesticidal activity against at least oneinsect pest, wherein said insect pest is the Soybean Cyst Nematode. 20.The polypeptide of claim 13, wheprein said polypeptide is characterizedby pesticidal activity against at least one insect pest, wherein saidinsect pest is the Soybean Cyst Nematode.